Use of mva or mvadeltae3l as immunotherapeutic agents against solid tumors

ABSTRACT

The present disclosure relates to modified vaccinia Ankara (MVA) virus or MVAAE3L delivered intratumorally or systemically as an anticancer immunotherapeutic agent, alone, or in combination with one or more immune checkpoint blocking agents for the treatment of malignant solid tumors. Particular embodiments relate to mobilizing the host&#39;s immune system to mount an immune response against the tumor.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 62/149,484 filed Apr. 17, 2015. The entire disclosure of thisprovisional application is incorporated by reference herein in itsentirety.

GOVERNMENT SUPPORT

Work disclosed in the present disclosure may have been supported byGrant No. K08AI073736 and Grant No. R56 AI095692 awarded by the NationalInstitutes of Health. The U.S. Government may have rights in thisinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 15, 2016, isnamed 11000-005111-WO0_SL.txt and is 3,493 bytes in size.

FIELD OF THE INVENTION

The present disclosure relates generally to the fields of oncology,virology and immunotherapy. It concerns the use of poxviruses,specifically the highly attenuated modified vaccinia virus Ankara (MVA),and a recombinant modified vaccinia Ankara virus with deletion ofvaccinia virulence factor E3 (MVAΔE3L) as cancer immunotherapeuticagents as well as for the development of immunotherapeutic vectors. Theforegoing poxviruses can also be used in combination with immunecheckpoint blockade therapy.

BACKGROUND Immune System and Cancer

Numerous studies support the importance of the differential presence ofimmune system components in cancer progression (1) (Jochems et al., ExpBiol Med, 236(5): 567-579 (2011)). Clinical data suggest that highdensities of tumor-infiltrating lymphocytes are linked to improvedclinical outcome (2) (Mlecnik et al., Cancer Metastasis Rev.; 30: 5-12,(2011)). The correlation between a robust lymphocyte infiltration andpatient survival has been reported in various types of cancer, includingmelanoma, ovarian, head and neck, breast, urothelial, colorectal, lung,hepatocellular, gallbladder, and esophageal cancer (3) (Angell et al.,Current Opinion in Immunology, 25:1-7, (2013)). Tumor immune infiltratesinclude macrophages, dendritic cells (DC), mast cells, natural killer(NK) cells, naïve and memory lymphocytes, B cells and effector T cells(T lymphocytes), primarily responsible for the recognition of antigensexpressed by tumor cells and subsequent destruction of the tumor cellsby cytotoxic T cells.

Despite presentation of antigens by cancer cells and the presence ofimmune cells that could potentially react against tumor cells, in manycases the immune system does not get activated or is affirmativelysuppressed. Key to this phenomenon is the ability of tumors to protectthemselves from immune response by coercing cells of the immune systemto inhibit other cells of the immune system. Tumors develop a number ofimmunomodulatory mechanisms to evade antitumor immune responses. Forexample, tumor cells secrete immune inhibitory cytokines (such as TGF-β)or induce immune cells, such as CD4⁺ T regulatory cells and macrophages,in tumor lesions to secrete these cytokines. Tumors have also theability to bias CD4⁺ T cells to express the regulatory phenotype. Theoverall result is impaired T-cell responses and induction of apoptosisor reduced anti-tumor immune capacity of CD8⁺ cytotoxic T cells.Additionally, tumor-associated altered expression of MHC class I on thesurface of tumor cells makes them ‘invisible’ to the immune response (4)(Garrido et al. Cancer Immunol. Immunother. 59(10), 1601-1606 (2010)).Inhibition of antigen-presenting functions and dendritic cell (DC)additionally contributes to the evasion of anti-tumor immunity (5)(Gerlini et al. Am. J. Pathol. 165(6), 1853-1863 (2004)).

Moreover, the local immunosuppressive nature of the tumormicroenvironment, along with immune editing, can lead to the escape ofcancer cell subpopulations that do not express the target antigens.Thus, finding an approach that would promote the preservation and/orrestoration of anti-tumor activities of the immune system would be ofconsiderable therapeutic benefit.

Immune checkpoints have been implicated in the tumor-mediateddownregulation of anti-tumor immunity and used as therapeutic targets.It has been demonstrated that T cell dysfunction occurs concurrentlywith an induced expression of the inhibitory receptors, CTLA-4 andprogrammed death 1 polypeptide (PD-1), members of the CD28 familyreceptors. PD-1 is an inhibitory member of the CD28 family of receptorsthat in addition to PD-1 includes CD28, CTLA-4, ICOS and BTLA. However,while promise regarding the use of immunotherapy in the treatment ofmelanoma has been underscored by the clinical use and even regulatoryapproval of anti-CTLA-4 (ipilimumab) and anti-PD-1 drugs (for examplepembrolizumab and nivolumab) the response of patients to theseimmunotherapies has been limited. Recent clinical trials, focused onblocking these inhibitory signals in T cells (e.g., CTLA-4, PD-1, andthe ligand of PD-1 PD-L1), have shown that reversing T cell suppressionis critical for successful immunotherapy (6, 7) (Sharma et al., Science348(6230), 56-61 (2015); Topalian et al., Curr Opin Immunol. 24(2),202-217 (2012)). These observations highlight the need for developmentof novel therapeutic approaches for harnessing the immune system againstcancer.

Poxviruses

Poxviruses, such as engineered vaccinia viruses, are in the forefront asoncolytic therapy for metastatic cancers (8) (Kirn et al., Nature ReviewCancer 9, 64-71 (2009)). Vaccinia viruses are large DNA viruses, whichhave a rapid life cycle and efficient hematogenous spread to distanttissues (9) (Moss, In Fields Virology (Lippincott Williams & Wilkins,2007), pp. 2905-2946). Poxviruses are well-suited as vectors to expressmultiple transgenes in cancer cells and thus to enhance therapeuticefficacy (10) (Breitbach et al., Current pharmaceutical biotechnology13, 1768-1772 (2012)). Preclinical studies and clinical trials havedemonstrated efficacy of using oncolytic vaccinia viruses and otherpoxviruses for treatment of advanced cancers refractory to conventionaltherapy (11-13) (Park et al., Lacent Oncol 9, 533-542 (2008); Kim etal., PLoS Med 4, e353 (2007); Thorne et al., J Clin Invest 117,3350-3358 (2007)). Poxvirus-based oncolytic therapy has the advantage ofkilling cancer cells through the combination of cell lysis, apoptosis,and necrosis. It also triggers innate immune sensing pathway thatfacilitates the recruitment of immune cells to the tumors and thedevelopment of anti-tumor adaptive immune responses. The currentoncolytic vaccinia strains in clinical trials (JX-594, for example) usewild-type vaccinia with deletion of thymidine kinase to enhance tumorselectivity, and with expression of transgenes such as granulocytemacrophage colony stimulating factor (GM-CSF) to stimulate immuneresponses (10) (Breitbach et al., Curr Pharm Biotechnol 13, 1768-1772(2012)). Many studies have shown however that wild-type vaccinia hasimmune suppressive effects on antigen presenting cells (APCs) (14-17)(Engelmayer et al., J Immunol 163, 6762-6768 (1999); Jenne et al., Genetherapy 7, 1575-1583 (2000); P. Li et al., J Immunol 175, 6481-6488(2005); Deng et al., J Virol 80, 9977-9987 (2006)), and thus adds to theimmunosuppressive and immunoevasive effects of tumors themselves. Bycontrast, modified vaccinia virus Ankara (MVA), a highly attenuatedvaccinia stain has moderate immune activating effects (18, 19) (Drillienet al., J Gen Virol 85, 2167-75 (2004); Dai et al., PLoS Pathog 10(4),e1003989 (2014).

Modified vaccinia virus Ankara (MVA) is a highly attenuated vacciniastrain that is an important vaccine vector for infectious diseases andcancers. MVA was derived from vaccinia strain through more than 570passages in chicken embryonic fibroblasts. MVA has a 31-kb deletion ofthe parental vaccinia genome and is non-replicative in most of mammaliancells. MVA was used in more than 120,000 people during WHO-sponsoredsmallpox vaccination, and was shown to be very safe for human use.Because of its safety and its ability to express foreign antigens, MVAhas been investigated as a vaccine vector against HIV, tuberculosis,malaria, influenza, coronavirus, and CMV, as well as cancers (20-25)(Sutter et al., Current drug targets. Infectious disorders 3, 263-271(2003); Gomez et al., Curr Gene Ther 8, 97-120 (2008); Gomez et al.,Curr Gene Ther 11, 189-217 (2011); Goepfert et al., J Infect Dis 203,610-619 (2011); Wyatt et al., Virology 372, 260-272 (2008); Garcia etal., Vaccine 29, 8309-8316 (2011)).

The investigation of MVA as cancer therapeutics has so far been limitedto its use as a vaccine vector to express tumor antigens (26, 27)(Tagliamonte et al. Hum Vaccin Immunother 10, 3332-3346 (2014); Verardiet al., Hum Vaccin Immunother 8, 961-970 (2012)). Various tumor antigenshave been expressed by MVA-based vectors, and some recombinant virusesare in various stages of clinical trials. For example, MVA-PSA-PAPexpresses both prostate specific antigen (PSA) and prostate acidphosphatase (PAP) is in clinical trials for patients with metastaticprostate cancer. The recombinant virus MVA-brachyury-TRICOM expressingtumor antigen brachyury and T cell co-stimulatory molecules is also inclinical trials for patients with metastatic cancers. The recombinantvirus MVA-p53 expressing p53 tumor suppressor, also in clinical trials,has been shown to be safe. Other tumor antigens that have been targetedinclude Her2, hMUC-1, TWIST, etc.

Although MVA is highly attenuated and moderately immunostimulatory, itretains multiple immune suppressive viral genes, including a keyvirulence factor, E3. MVAΔE3L, a recombinant MVA virus furtherattenuated by deletion of the vaccinia virulent factor E3, is unable toreplicate in primary chicken embryo fibroblasts (CEFs), but retains itsreplication capacity in baby hamster kidney BHK-21 cells (28) (Hornemannet al., J Virol 77(15), 8394-07 (2003). MVAΔE3L is capable ofreplicating viral DNA genomes in CEFs and is deficient in viral lateprotein synthesis (28) (Hornemann et al., J Virol 77(15), 8394-07(2003). It also induces apoptosis in CEF (28) (Hornemann et al., J Virol77(15), 8394-07 (2003)). MVAΔE3L infection of HeLa cells had similareffects, with impaired viral replication, viral late gene transcriptionand translation (29) (Ludwig et al., J Virol 79(4), 2584-2596 (2005)).MVAΔE3L also induces apoptosis in HeLa cells, possibly throughactivating the mitochondrial pathway (29) (Ludwig et al., J Virol 79(4),2584-2596 (2005)). dsRNA are produced during intermediate genetranscription, which can lead to the activation of 2′-5′-oligoadenylatesynthase/RNase L and Protein Kinase R (PKR). In PKR-deficient MEFs,MVAΔE3L gains its ability to express intermediate and late proteins((29) (Ludwig et al., J Virol 79(4), 2584-2596 (2005)).

One study suggests that pro-apoptotic protein Noxa plays a role inMVAΔE3L apoptosis induction (30) (Fischer et al., Cell Death Differ 13,109-118 (2006)). Although an early study showed that MVAΔE3L induceshigher levels of type I IFN in CEFs than MVA, the exact mechanism wasnot fully elucidated (28) (Hornemann et al., J Virol 77(15), 8394-07(2003).

One MVAΔE3L has been described in U.S. Pat. No. 7,049,145 incorporatedby reference. It is infection competent but nonreplicative in mostmammalian cells including mouse and human.

This disclosure focuses on the intratumoral delivery of MVA or MVAΔE3Las anticancer immunotherapeutic agents. It was hoped that intratumoraldelivery of MVA or MVAΔE3L would elicit innate immune responses fromtumor infiltrating immune cells (e.g. leukocytes), tumor cells, andtumor associated stromal cells, and lead to induction of type I IFN andproinflammatory cytokines and chemokines, which would result in thealteration of the tumor immune suppressive microenvironment.

The recent discovery of tumor neoantigens in various solid tumorsindicates that solid tumors harbor unique neoantigens that usuallydiffer from person to person (31, 32) (Castle et al., Cancer Res 72,1081-1091 (2012); Schumacher et al., Science 348, 69-74 (2015) Therecombinant viruses disclosed in this invention do not work byexpressing tumor antigens. Intratumoral delivery of the presentrecombinant MVA viruses allows efficient cross-presentation of tumorneoantigens and generation of anti-tumor adaptive immunity within thetumors (and also extending systemically), and therefore lead to “in situcancer vaccination” utilizing tumor differentiation antigens andneoantigens expressed by the tumor cells in mounting an immune responseagainst the tumor.

Despite the presence of neoantigens generated by somatic mutationswithin tumors, the functions of tumor antigen-specific T cells are oftenheld in check by multiple inhibitory mechanisms (33) (Mellman et al.,Nature 480, 480-489 (2011)). For example, the up-regulation of cytotoxicT lymphocyte antigen 4 (CTLA-4) on activated T cells can compete with Tcell co-stimulator CD28 to interact with CD80 (B71)/CD86 (B7.2) ondendritic cells (DCs), and thereby inhibit T cell activation andproliferation. CTLA-4 is also expressed on regulatory T (Treg) cells andplays an important role in mediating the inhibitory function of Tregs_((34, 35)) (Wing et al., Science 322, 271-275 (2008); Peggs, et al., JExp Med 206, 1717-1725 (2009)). In addition, the expression ofPD-L/PD-L2 on tumor cells can lead to the activation of the inhibitoryreceptor of the CD28 family, PD-1, leading to T cell exhaustion.Immunotherapy utilizing antibodies against inhibitory receptors, such asCTLA-4 and programmed death 1 polypeptide (PD-1), have shown remarkablepreclinical activities in animal studies and clinical responses inpatients with metastatic cancers, and have been approved by the FDA forthe treatment of metastatic melanoma, non-small cell lung cancer, aswell as renal cell carcinoma (6, 36-39)(Leach et al., Science 271,1734-1746 (1996); Hodi et al., NEJM 363, 711-723 (2010); Robert et al.,NEJM 364, 2517-2526 (2011); Topalian et al., Cancer Cell 27, 450-461(2012); Sharma et al., Science 348(6230), 56-61 (2015))

Melanoma

Melanoma, one of the deadliest cancers, is the fastest growing cancer inthe US and worldwide. Its incidence has increased by 50% among youngCaucasian women since 1980, primarily due to excess sun exposure and theuse of tanning beds. According to the American Cancer Society,approximately 78,000 people in the US will be diagnosed with melanoma in2015 and almost 10,000 people (or one person per hour) will die frommelanoma. In most cases, advanced melanoma is resistant to conventionaltherapies, including chemotherapy and radiation. As a result, peoplewith metastatic melanoma have a very poor prognosis, with a lifeexpectancy of only 6 to 10 months. The discovery that about 50% ofmelanomas have mutations in BRAF (a key tumor-promoting gene) opened thedoor for targeted therapy in this disease. Early clinical trials withBRAF inhibitors showed remarkable, but unfortunately not sustainable,responses in patients with melanomas with BRAF mutations. Therefore,alternative treatment strategies for these patients, as well as otherswith melanoma without BRAF mutations, are urgently needed.

Human pathological data indicate that the presence of T-cell infiltrateswithin melanoma lesions correlates positively with longer patientsurvival (40) (Oble et al. Cancer Immun. 9, 3 (2009)). The importance ofthe immune system in protection against melanoma is further supported bypartial success of immunotherapies, such as the immune activatorsIFN-α2b and IL-2 (41) (Lacy et al. Expert Rev Dermatol 7(1):51-68(2012)) as well as the unprecedented clinical responses of patients withmetastatic melanoma to immune checkpoint therapy, including anti-CTLA-4and anti-PD-1/PD-L1 either agent alone or in combination therapy (6, 7,37, 42-45) (Sharma and Allison, Science 348(6230), 56-61 (2015); Hodi etal., NEJM 363(8), 711-723 (2010); Wolchok et al., Lancet Oncol. 11(6),155-164 (2010); Topalian et al., NEJM 366(26), 2443-2454 (2012); Wolchoket al., NEJM 369(2), 122-133 (2013); Hamid et al., NEJM 369(2), 134-144(2013); Tumeh et al., Nature 515(7528), 568-571 (2014). However, manypatients fail to respond to immune checkpoint blockade therapy alone.The addition of virotherapy might overcome resistance to immunecheckpoint blockade, which is supported by animal tumor models (46)(Zamarin et al., Sci Transl Med 6(226), 2014).

Type I IFN and the Cytosolic DNA-Sensing Pathway in Tumor Immunity.

Type I IFN plays important roles in host antitumor immunity (47)(Fuertes et al., Trends Immunol 34, 67-73 (2013)). IFNAR1-deficient miceare more susceptible to develop tumors after implantation of tumorcells; Spontaneous tumor-specific T cell priming is also defective inIFNAR1-deficient mice (48, 49) (Diamond et al., J Exp Med 208, 1989-2003(2011); Fuertes et al., J Exp Med 208, 2005-2016 (2011)). More recentstudies have shown that the cytosolic DNA-sensing pathway is importantin the innate immune sensing of tumor-derived DNA, which leads to thedevelopment of antitumor CD8⁺ T cell immunity (50) (Woo et al., Immunity41, 830-842 (2014)). This pathway also plays a role in radiation-inducedantitumor immunity (51) (Deng et al., Immunity 41, 843-852 (2014)).Although spontaneous anti-tumor T cell responses can be detected inpatients with cancers, cancers eventually overcome host antitumorimmunity in most patients. Novel strategies to alter the tumor immunesuppressive microenvironment would be beneficial for cancer therapy.

SUMMARY

The present disclosure relates to the discovery that both MVA andMVAΔE3L have properties that can be used effectively in developingimmunotherapies against cancers. Intratumoral injection of MVA orMVAΔE3L leads to tumor regression and even eradication, and to thegeneration of systemic antitumoral immunity. Therefore, both MVA andMVAΔE3L can be used as immunotherapy for the treatment of solid tumors.Moreover, the combination of intratumoral delivery of MVA-basedvirotherapy and immune checkpoint blockade (or checkpoint agonisttherapy), delivered either systemically or intratumorally, isanticipated to lead to enhanced antitumoral activities in injectedtumors as well as non-injected distant tumors.

The present inventors observed that MVA infection of conventionaldendritic cells (cDCs) triggers type I IFN via the cytosolic DNA-sensingpathway mediated by the newly discovered cytosolic DNA sensor cGAS(cyclic GMP-AMP synthase) and its adaptor STING (stimulator of IFNgenes). By contrast, wild-type vaccinia infection of cDCs fails toinduce type I IFN. They also observed that a recombinant MVA virus withdeletion of vaccinia virulence factor E3 (MVAΔE3L) infection of cDCsinduces higher levels of type I IFN than MVA. It also activates theinnate immune-sensing pathways for MVAΔE3L virus in cDCs, and inducestype I IFN, inflammatory cytokines and chemokines, and apoptosis incancer cells by MVA and MVAΔE3L.

These observations lead to the possibility of using these highlyattenuated modified vaccinia viruses as immune activators to alter thetumor-induced immune suppressive microenvironment through induction oftype I IFN and other inflammatory cytokines and chemokines in tumorcells as well as in immune cells (in other words, to induce antitumorimmune responses in the host or to enhance antitumor responses that mayalready be ongoing and to reverse their suppression). This in turn leadsto more efficient tumor antigen presentation, and to the generation andactivation of anti-tumor cytotoxic CD8⁺ T cells, effector CD4⁺ T cells,as well as the reduction of immune suppressive CD4⁺ regulatory T cellsand tumor-associated macrophages. Because MVA and MVAΔE3L are safevaccine vectors the use of such viral vectors within the tumor allowstumor antigen release, efficient presentation, and the generation ofantitumor effector and memory T cell responses and antitumor antibodyproduction. Indeed, the inventors observed that MVA and MVAΔE3L usedintratumorally lead to activation of dendritic cells and improvedpresentation of tumor antigens (including oncogenic viral antigens,tumor differentiation antigens, and tumor neoantigens).

The localized (e.g., intratumoral) injection of MVA and MVAΔE3L can beused for various stages of tumors. For early stage cancer, virotherapycan be used 2-3 weeks prior to surgical removal of the tumor. Duringthat time frame, the host would have developed systemic anti-tumoradaptive immunity. For advanced cancer, virotherapy can be used incombination with other treatment modalities, including surgery,chemotherapy, targeted therapy, radiation, and immune checkpointtherapy, which will be detailed below.

Based on results obtained by the present inventors with inactivated MVAand described in PCT US2016/019663 filed Feb. 25, 2016, incorporated byreference in its entirety for all purposes, the present inventorshypothesize that intratumoral injection of MVA or MVAΔE3L would provideadditional beneficial effects to a PD-1 or CTLA-4 targeting approach,through induction of type I IFN in immune cells and cancer cells,altering the tumor immune suppressive environment via the activation ofimmune cells including dendritic cells as well as facilitating tumorantigen presentation.

In one aspect, the disclosure is directed to a method for treating asolid malignant tumor in a subject comprising delivering to tumor cellsof the subject an amount of MVA or MVAΔE3L effective to induce theimmune system of the subject to mount an immune response against thetumor, for example as set forth above in this Summary so as toaccomplish one or more of the following (regardless of order): reducethe size of the tumor, eradicate the tumor, inhibit growth of the tumor,or inhibit metastasis or metastatic growth of the tumor.

In another aspect, the disclosure is directed to a method for treating amalignant tumor comprising:

-   -   delivering to tumor cells of the subject an amount of MVA or        MVAΔE3L effective to induce the immune system of the subject to        mount an immune response against the tumor.

In some embodiments one or more of the following specific features arealso present:

-   -   the recruitment and activation of effector T cells is        accompanied by a reduction of regulatory CD4⁺ cells in the        tumor;    -   the tumor is melanoma or colon carcinoma;    -   a regimen of periodic delivery of MVA or MVAΔE3L is continued        until it induces tumor regression or eradication;    -   a regimen of periodic delivery of the MVA or MVAΔE3L is        continued for several weeks, months or years or indefinitely as        long as benefits persist;    -   a regimen of periodic delivery of the MVA or MVAΔE3L is        continued indefinitely until the maximum tolerated dose is        reached;    -   delivery of the MVA or MVAΔE3L is by parenteral injection;    -   delivery of the MVA or MVAΔE3L is by intratumoral injection;    -   delivery of the MVA or MVAΔE3L is by intravenous injection;    -   the subject is a human;    -   the MVA or MVAΔE3L is delivered at a dosage per administration        within the range of about 10⁵-10¹⁰ plaque-forming units (pfu);    -   the MVA or MVAΔE3L is delivered at a dosage per administration        within the range of about 10⁶ to about 10⁹ plaque-forming units        (pfu);    -   the amount delivered is sufficient to infect all tumor cells;    -   the delivery is repeated with a frequency within the range from        once per month to two times per week;    -   the treatment continues for a period of weeks, months or years;    -   the delivery is repeated with a frequency within the range from        once per month to two times per week;    -   the melanoma is metastatic melanoma.

Delivery of MVA or MVAΔE3L in the locale of the tumor induces the immunesystem of a subject afflicted with a malignant solid tumor to mount animmune response against the tumor. Stimulation of the subject's immunesystem against the tumor can be manifest (and may indeed be tested) byone or more of the following immunological effects:

-   -   an increase in antitumor cytotoxic CD8⁺ and effector CD4⁺ T        cells within the tumor and/or in tumor-draining lymph nodes;    -   induction of maturation of dendritic cells infiltrating said        tumor through induction of type I IFN;    -   induction of activated antitumor effector T cells in the subject        recognizing tumor cells within the tumor and/or in tumor        draining lymph nodes;    -   reduction of immune suppressive (regulatory) CD4⁺ T cells within        the tumor; and    -   induction of cells of the tumor to express MHC Class I on their        surface and to produce Type I IFN.

More particularly, in one aspect, the present disclosure is directed toa method for treating a subject afflicted with a malignant solid tumorr, the method comprising delivering to the cells of the tumor a modifiedvaccinia virus selected from the group of MVA and MVAΔE3Landcombinations thereof and thereby treating the tumor.

In some embodiments, the amount of said virus is effective to bringabout one or more of the following:

-   -   a. induce the immune system of the subject to mount an immune        response against the tumor or enhance an ongoing response by the        immune system against the tumor;    -   b. reduce the size of the tumor;    -   c. eradicate the tumor;    -   d. inhibit growth of the tumor;    -   e. inhibit metastasis of the tumor; and    -   f. reduce or eradicate metastatic tumor.

In another aspect the disclosure provides a method for treating a solidmalignant tumor in a subject comprising delivering to tumor cells of thesubject an amount of MVA or MVAΔE3L or a combination thereof effectiveto induce the immune system of the subject to mount an immune responseagainst the tumor or to enhance an ongoing immune response of saidsubject against the tumor, so as to accomplish one or more of thefollowing: reduce the size of the tumor, eradicate the tumor, inhibitgrowth of the tumor, inhibit metastatic growth of the tumor, induceapoptosis of tumor cells or prolong survival of the subject.

In another aspect, the present disclosure is directed to A method fortreating a solid malignant tumor in a subject comprising delivering to atumor of the subject an amount of modified vaccinia virus Ankara (MVA)or MVAΔE3L or a combination of both effective to bring about at leastone of the following immunologic effects:

-   -   a. increase at least one of effector CD8⁺ T cells and effector        CD4⁺ T cells within the tumor and/or in tumor-draining lymph        nodes;    -   b. induce maturation of dendritic cells infiltrating said tumor        through induction of type I IFN;    -   c. reduce immune suppressive (regulatory) CD4⁺ T cells within        the tumor;    -   d. reduce immune suppressive tumor-associated macrophages (TAM)        within the tumor;    -   e. induce type I IFN, inflammatory cytokine and chemokine        production in immune cells and stromal fibroblasts.    -   f.

In some embodiments of each of the foregoing aspects:

-   -   the MVA or MVAΔE3L is not harboring nucleic acid encoding or        expressing a tumor antigen;    -   the tumor includes tumor located at the site of MVA or MVAΔE3L        or tumor located elsewhere in the body of the subject;    -   the recruitment and activation of CD4⁺ effector T cells is        accompanied by a reduction of regulatory CD4⁺ cells in said        tumor.    -   the tumor is melanoma or colon carcinoma or another solid tumor;    -   delivery of the MVA or MVAΔE3L is continued until it induces        tumor regression or eradication;    -   delivery of the MVA or MVAΔE3L is continued for several weeks,        months or years or indefinitely as long as benefits persist or a        maximum tolerated dose is reached;    -   delivery of the MVA or MVAΔE3L is continued indefinitely until        the maximum tolerated dose is reached;    -   delivery of the MVA or MVAΔE3L is by parenteral, e.g.,        intratumoral or intravenous injection;    -   the subject is a human;    -   MVA or MVAΔE3L is delivered at a dosage per administration        within the range of about 10⁵-10¹⁰ plaque-forming units (pfu);    -   the MVA or MVAΔE3L is delivered at a dosage per administration        within the range of about 10⁶ to about 10⁹ plaque-forming units        (pfu);    -   the amount delivered is sufficient to infect all tumor cells;    -   the delivery is repeated with a frequency within the range from        once per month to two times per week;    -   the delivery is repeated once weekly;    -   the melanoma is metastatic melanoma;    -   the MVA is MVAΔE3L;

In still another aspect, the present disclosure provides a method fortreating a malignant tumor in a subject, the method comprisingdelivering to tumor cells of the subject a virus selected from the groupconsisting of modified vaccinia Ankara (MVA), MVAΔE3L and a combinationthereof in an amount effective to induce the immune system of thesubject to ount an immune response against the tumor or to enhance anongoing immune response f said subject against the tumor and conjointlyadministering to the subject a second amount of an immune checkpointblocking agent or an immune checkpoint agonist effective to block immunesuppressive mechanisms within the tumor.

In more specific embodiments:

-   -   the immune suppressive mechanisms are elicited by tumor cells,        stromal cells, or tumor infiltrating immune cells;    -   the administration is by parenteral route;    -   the delivery is by intratumoral injection and the administration        is by intravenous route;    -   both the delivery and the administration are by intravenous        route;    -   both the delivery and the administration are by intratumoral        injection;    -   the immune checkpoint blocking agent is selected from the group        consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA4        inhibitors, inhibitory antibodies against LAG-3 (lymphocyte        activation gene 3), TIM3 (T cell Immunoglobulin and Mucin-3),        B7-H3, and TIGIT (T-cell immunoreceptor with Ig and ITIM        domains); and the immune checkpoint agonist is selected from the        group consisting of anti-ICOS antibody anti-OX40 antibody        agonist antibody against 4-1BB (CD137) and against GITR;    -   any one of said inhibitors or agonists is an antibody;    -   the tumor is primary or metastatic melanoma or primary or        metastatic colon carcinoma or another solid tumor.    -   the virus is delivered and the immune checkpoint blocking agent        is administered each according to its own administration        schedule of spaced apart intervals;    -   a first dose of the virus is delivered first and after a lapse        of time a first dose of the immune checkpoint blocking agent is        administered;    -   the delivery and administration occur in parallel during the        same overall period of time;    -   one or both of the virus and the immune checkpoint blocking        agent are respectively delivered and administered during a        period of time of several weeks, months or years, or        indefinitely as long as benefits persist and a maximum tolerated        dose is not reached;    -   the virus is delivered at a dosage per administration within the        range of about 10⁵-10¹⁰ plaque-forming units (pfu);    -   the virus is delivered at a dosage per administration within the        range of about 10⁶ to about 10⁹ plaque-forming units (pfu);    -   the virus delivery is repeated with a frequency within the range        from once per month to two times per week;    -   the virus delivery is repeated once weekly;    -   the virus is MVAΔE3L;    -   the subject is a human;    -   the virus is MVA;    -   the virus and the immune checkpoint blocking agent or agonist        are administered simultaneously;    -   the virus and the immune checkpoint blocking agent or agonist        are administered in the same composition;    -   the MVA and the immune checkpoint blocking agent are delivered        intratumorally;    -   the virus and the immune checkpoint blocking agent are        administered sequentially;    -   the inactivated MVA and the immune checkpoint blocking agent are        delivered intratumorally.

In an additional aspect, the present disclosure provides a compositionfor use in treating a solid tumor comprising an amount of a modifiedvaccinia virus selected from the group consisting of MVA and MVAΔE3L andcombinations thereof effective to induce the immune system of a host towhom said composition will be administered to mount an immune responseagainst the tumor or to enhance an ongoing immune response of the hostagainst the tumor; and a pharmaceutically acceptable carrier or diluent.

In more specific embodiments of the composition the effective amount iswithin the range of about 10⁵-10¹⁰ plaque-forming units (pfu) in a unitdosage form; or the effective amount is within the range of 10⁶ to 10⁹pfu.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphical representations of data showing that MVAinduces type I IFN production in murine cDCs. FIG. 1A are graphs showingsecretion levels of IFN-α and IFN-β in GM-CSF-BMDCs at 1, 4, 8, 14, and22 hours post infection with WT VAC or MVA. FIG. 1B are bar graphsshowing mRNA levels of IFNA4 and IFNB in GM-CSF-BMDCs at 6 hours postinfection with WT VAC or MVA.

FIG. 2 is a series of bar graphs showing that transcription factorsIRF3/IRF7 and the type I IFN positive feedback loop mediated by IFNAR1are required for the induction of type I IFN in murine cDCs by MVA.FIGS. 2A-2C are bar graphs of IFN-α and IFN-β concentrations inGM-CSF-BMDCs generated from IRF3^(−/−) (2A), IRF7−/− (2B), IFNAR1^(−/−)(2C) mice, or their age-matched WT controls. Data are means±SEM (n=3). Arepresentative experiment is shown, repeated twice. *, p<0.05; **,p<0.01; ***, p<0.001.

FIG. 3 is a series of graphical representations showing that STING isrequired for the induction of type I IFN and IRF3 phosphorylation by MVAin BMDCs. FIG. 3A shows bar graphs of IFN-α and IFN-β secretion levelsin GM-CSF-BMDCs cell generated from Sting^(+/+) and Sting^(Gt/Gt) mice,stimulated with LPS or infected with MVA. FIG. 3B shows mRNA expressionlevels of IFNA4 and IFNB in GM-CSF-BMDCs cell generated from Sting^(+/+)and Sting^(Gt/Gt) mice and infected with MVA. FIG. 3C is a scanned imageof immunoblot showing the proteins levels of phospho-TBK1, TBK1,phosphoserine-396 of IRF3, IRF3, and GAPDH. “hpi”, hours post infection,“M”, mock infection control. FIG. 3D are bar graphs showing thesecretion levels of IFN-α and IFN-β in Sting^(Gt/Gt), IRF3^(−/−) andage-matched WT C57B/6 control mice infected with MVA. Data are means±SD.Results shown are representative of two independent experiments.

FIG. 4 is a series of graphical representations demonstrating that cGASis the critical cytosolic DNA sensor for MVA infection of cDCs. FIG. 4Aare bar graphs showing IFN-α and IFN-β secretion levels in GM-CSF-BMDCsgenerated from cGAS^(−/−) mice and its age-matched WT controls, andinfected with MVA. Data are means±SEM (n=3). A representative experimentis shown, repeated twice (***, p<0.001). FIG. 4B are bar graphs showingmRNA expression levels of IFNA4 and IFNB in GM-CSF-BMDCs cell generatedfrom cGAS^(−/−) mice and its age-matched WT controls, and infected withMVA. Data are means±SEM (n=3). A representative experiment is shown,repeated twice (***, p<0.001). FIG. 4C is a scanned image of immunoblotshowing the protein levels of phospho-TBK1, TBK1, phosphoserine-396 ofIRF3, IRF3, and GAPDH in cGAS^(+/+) and cGAS^(−/−) cDCs infected withMVA. “hpi”, hours post infection.

FIG. 5 is a series of graphical representations showing that MVAΔE3Linduces higher levels of type I IFN gene expression in BMDCs than MVAdoes. FIG. 5A is a scanned image of an immunoblot showing protein levelsof E3 and β-actin in GM-CSF-BMDCs infected with WT VAC, MVA, or MVAΔE3L.“hpi,” hours post infection, “M”, mock infection control. FIG. 5B arebar graphs showing mRNA levels of IFNA4 and IFNB in GM-CSF-BMDCsinfected with MVA or with MVAΔE3L. Data are means±SEM (n=3). Arepresentative experiment is shown, repeated twice. ***, p<0.001;comparisons were made between MVA and MVAΔE3L infected cells. FIG. 5Care bar graphs showing mRNA levels of IFNA4 and IFNB in GM-CSF-BMDCsgenerated from IRF3^(−/−) mice and age-matched WT C57B/6 mice andinfected with MVA or with MVAΔE3L. Data are means±SEM (n=3). Arepresentative experiment is shown, repeated twice. ***, p<0.001;comparisons were made between MVA and MVAΔE3L infected cells. FIG. 5D isa scanned image of an immunoblot showing protein levels of p-IRF3 andβ-actin in GM-CSF-BMDCs infected with MVA or with MVAΔE3L.

FIG. 6 is a series of bar graphs showing that cGAS is required for theinduction of type I IFN by MVAΔE3L in cDCs. FIG. 6A includes bar graphsshowing mRNA levels of IFNA4 and IFNB in cGAS^(+/+) and cGAS^(−/−) cDCsinfected with MVAΔE3L. FIG. 6B includes bar graphs showing IFN-α andIFN-β secretion levels in cGAS^(+/+) and cGAS^(−/−) cDCs infected withMVAΔE3L or treated with cGAMP, an agonist for STING. FIG. 6C is ascanned image of an immunoblot showing protein levels of p-IRF3 andGAPDH in cGAS^(+/+) and cGAS^(−/−) cDCs infected with MVAΔE3L.

FIG. 7 is a series of scanned images of Western blot data showing thatdsRNA-sensing pathway also plays a role in MVAΔE3L-inducedphosphorylation of IRF3. FIG. 1A shows protein levels of p-IRF3 andβ-actin in cDCs generated from WT, STING^(Gt/Gt), or MAVS^(−/−) mice,and infected with MVAΔE3L or not treated (NT). “hpi”, hours postinfection. FIG. 7B shows protein levels of phospho-TBK1, TBK1,phosphoserine-396 of IRF3, IRF3, and GAPDH in cDCs generated from WT orSTING^(Gt/Gt)/MDA5^(−/−) (DKO) mice, and infected with MVAΔE3L or MVA.“hpi”, hours post infection.

FIG. 8 is a series of bar graphs showing that MVA and MVAΔE3L infectionof murine primary fibroblasts leads to induction of gene expression ofIfnb (FIG. 8A), Ccl4 (FIG. 8B), Il6 (FIG. 8C), and Ccl5 (FIG. 8D), whichis largely dependent on cGAS. “NT”, not treated. MVAΔE3L-inducedexpression of Ifnb (FIG. 8E), Ccl4 (FIG. 8F), Il6 (FIG. 8G), and Ccl5(FIG. 8H) is completely abolished in STING and MDA5-double deficientmurine primary fibroblasts.

FIG. 9 is a series of bar graphs showing that MVA and MVAΔE3L infectionof murine primary fibroblasts leads to production of IFN-β (FIG. 9A),CCL4 (FIG. 9B), IL-6 (FIG. 9C), and CCL5 (FIG. 9D), which is largelydependent on STING and with some contribution from MDA5.

FIG. 10 is a series of bar graphs showing that MVAΔE3L infection leadsto higher secretion levels of Ifna4 (FIG. 10A), Ifnb (FIG. 10B), Il6(FIG. 10C), Tnf (FIG. 10D), Ccl4 (FIG. 10E), and Ccl5 (FIG. 10F) thanMVA in B16-F10 melanoma cells. “NT”, not treated.

FIG. 11 is a series of scanned immunoblot images showing that infectionof B16-F10 melanoma cells with MVA or MVAΔE3L induces apoptosis. FIG.11A shows protein levels of PARP, cleaved PARP, and β-actin in B16-F10melanoma cells infected with MVA or MVAΔE3L. FIG. 11B shows proteinlevels of MCL-1, and β-actin in B16-F10 melanoma cells infected with MVAor MVAΔE3L. “hpi”, hours post infection. FIG. 11C shows levels ofphosphorylated IRF3 and GAPDH in B16-F10 melanoma cells infected withMVA or MVAΔE3L. “hpi”, hours post infection.

FIG. 12 is a series of graphs showing that intratumoral injection of MVAand MVAΔE3L leads to prolonged survival of tumor-bearing mice anderadication of tumors in some mice. FIGS. 12A-C are graphs of tumorvolume over time in individual mice injected with PBS (A), MVA (B), andMVAΔE3L (C). FIG. 12D is a Kaplan-Meier survival curve of tumor-bearingmice injected with PBS, MVA, or MVAΔE3L. ****, p<0.0001 (MVA vs. PBSgroup); ***, p<0.001 (MVAΔE3L vs. PBS group). FIG. 12E is a Kaplan-Meiersurvival curve of tumor-free mice after successful treatment with MVA orMVAΔE3L, and challenged with B16-F10 melanoma cells at the contralateralside. Naïve mice have never received any tumor cells or viruses in thepast.

FIG. 13 is a series of graphical representations of data showing thatintratumoral injection with MVA leads to immunological changes in thetumor microenvironment. FIG. 13A-B are dot-plots of flow cytometricanalysis of CD4⁺ cells expressing FoxP3 in tumors treated with eitherPBS (13A) or MVA (13B). FIGS. 13D-E are dot-plots of flow cytometricanalysis of CD8⁺ cells expressing Granzyme B in tumors treated witheither PBS (13D) or MVA (13E). FIGS. 13G-H are scatterplots of flowcytometric analysis of CD8⁺ cells expressing Ki-67 in tumors treatedwith either PBS (13G) or MVA (13H). FIG. 13C is a graph depictingpercentages of CD4⁺Foxp3⁺ in tumors treated with PBS or MVA. FIG. 13F isa graph depicting percentages of Granzyme B⁺CD8⁺ cells in tumors treatedwith PBS or MVA. FIG. 13 I is a graph depicting percentages ofCD8⁺Ki-67⁺ in tumors treated with PBS or MVA.

FIG. 14 is a series of graphical representations of data showing thatintratumoral injection with MVA induces immunological changes in thetumor draining lymph nodes (TDLNs). FIGS. 14A-B are dot-plots of flowcytometric analysis of Granzyme B⁺CD8⁺ cells in TDLNs of PBS (14A) orMVA (14B) treated mice. FIGS. 14C-D are dot-plots of flow cytometricanalysis of Ki-67⁺CD8⁺ cells in TDLNs of PBS (14D) or MVA (14E) treatedmice. FIG. 14C is a graph depicting percentages of Granzyme B⁺CD8⁺ cellsin TDLNs from mice treated with PBS or MVA. FIG. 14F is a graphdepicting percentages of CD8⁺Ki-67⁺ in TDLNs from mice treated with PBSor MVA.

FIG. 15 is a series of graphic representations showing that MVAΔE3Linduces type I IFN and inflammatory cytokines/chemokines production inMC38 colon cancer cells. FIGS. 15A-D are bar graphs showing proteinlevels of IFN-β (15A), IL-6 (15B), CCL4 (15C), and CCL5 (15D) in thesupernatants of MC38 colon cancer cells infected with MVA or MVAΔE3L.FIGS. 15E-H are bar graphs showing the mRNA levels of Ifnb (15E), Il6(15F), Ccl4 (15G), and Ccl5 (15H) in MC38 colon cancer cells at 6 h postinfection with MVA or MVAΔE3L. FIG. 15I is a scanned image of a Westernblot showing protein levels of PARP, cleaved-PARP, phosphor-IRF-3, IRF3,and β-actin. “hpi”, hours post infection.

FIG. 16 is a series of graphs showing that MVAΔE3L inhibitstumorigenesis in murine model of colon carcinoma. FIGS. 16A and 16B areplots of tumor volume v. time from injection of PBS or virus in miceshowing that intratumoral injection of MVAΔE3L is effective for thetreatment of murine colon adenocarcinoma (MC38 cells) implantedunilaterally in mice (C57B/6). The tumor volumes of mice treated withPBS or MVAΔE3L groups prior to treatment (day 0) and up to 45 dayspost-treatment are shown. FIG. 16C is a Kaplan-Meier survival curve forthe treated mice (MVAΔE3L) versus control mice (PBS). ***, p<0.001(MVAΔE3L vs. PBS group).

FIG. 17 is a series of graphical representations of data showing thatintratumoral injection of MVA or MVAΔE3L induced antitumor effects innon-injected distant tumors in a murine B16-F10 melanoma bilateralimplantation model. FIG. 17A-F are graphs of injected (A, C, E) andnon-injected (B, D, F) tumor volume plotted against time (days) afterPBS, MVA, or MVAΔE3L injection respectively. FIG. 17G is a Kaplan-Meiersurvival curve of tumor-bearing mice (B16-F10 cells) injected with PBS(filled circles), MVA (filled squares), or MVAΔE3L (filled triangles).****, p<0.0001 (MVAΔE3L vs. PBS group); ***, p<0.001 (MVA vs. PBSgroup).

FIG. 18 is a series of graphical representations of data showing thatintratumoral injection of MVA or MVAΔE3L induces activated effector CD8⁺and CD4⁺ T cells and reduces regulator CD4⁺ T cells in both injected andnon-injected tumors in a murine B16-F10 melanoma bilateral implantationmodel. FIG. 18A are dot-plots of flow cytometric analysis of CD3⁺CD8⁺ Tcells in both injected and non-injected tumors treated with PBS, MVA orMVAΔE3L. FIG. 18B is a graph of % CD3⁺CD8⁺ T cells in both injected andnon-injected tumors treated with PBS, MVA or MVAΔE3L. FIGS. 18C and Eare dot-plots of flow cytometric analysis of CD8⁺ cells expressingGranzyme B⁺ (18C) or Ki-67 (18E). FIGS. 18D and F are graphs of %CD8⁺Granzyme B⁺ (18D), CD8⁺Ki-67⁺ (18F) T cells in both injected andnon-injected tumors treated with PBS, MVA or MVAΔE3L. FIG. 18G aredot-plots of flow cytometric analysis of CD4⁺Foxp3⁺ T cells in bothinjected and non-injected tumors treated with PBS, MVA or MVAΔE3L. FIG.18H is a graph of % CD4⁺Foxp3⁺ T cells in both injected and non-injectedtumors treated with PBS, MVA or MVAΔE3L. FIGS. 18I and K are dot-plotsof flow cytometric analysis of CD4⁺ cells expressing Granzyme B⁺ (18I)or Ki-67 (18K). FIGS. 18J and L are graphs of % CD4⁺Granzyme B⁺ (18J),CD8⁺Ki-67⁺ (18L) T cells in both injected and non-injected tumorstreated with PBS, MVA or MVAΔE3L. (*, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001).

FIG. 19 is a series of graphical representations of data showing thatintratumoral injection of MVA or MVAΔE3L reduces tumor-associatedmacrophages (TAMs) in a murine B16-F10 melanoma model. FIG. 19A aredot-plots of flow cytometric analysis of TAM cells(CD45⁺Ly6C⁻MHCII⁺CD24^(lo)F4/80⁺CD11b⁺CD11c⁺) in tumors treated withPBS, MVA or MVAΔE3L. FIG. 19B-D are graphs of % TAM, TAM1(CD11C^(lo)CD11b^(hi)), and TAM2 (CD11C^(hi)CD11b^(lo) in CD45⁺ cells intumors treated with PBS, MVA or MVAΔE3L. (*, p<0.05; ns:non-significant).

DETAILED DESCRIPTION Definitions

As used herein the following terms shall have the meanings ascribed tothem below unless the context clearly indicates otherwise:

-   -   “Cancer” refers to a class of diseases of humans and animals        characterized by uncontrolled cellular growth. Unless otherwise        explicitly indicated, the term “cancer” may be used herein        interchangeably with the terms “tumor,” “malignancy,”        “hyperproliferation” and “neoplasm(s);” the term “cancer        cell(s)” is interchangeable with the terms “tumor cell(s),”        “malignant cell(s),” “hyperproliferative cell(s),” and        “neoplastic cell(s)”.    -   “Melanoma” refers to a malignant neoplasm originating from cells        that are capable of producing melanin. The term melanoma is        synonymous with “malignant melanoma”. Melanoma metastasizes        widely, involving a patient's lymph nodes, skin, liver, lungs        and brain tissues.    -   “Solid tumor” refers to all neoplastic cell growth and        proliferation, and all pre-cancerous and cancerous cells and        tissues, except for hematologic cancers such as lymphomas,        leukemias and multiple myeloma. Examples of solid tumors        include, but are not limited to: soft tissue sarcoma, such as        fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,        osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,        lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,        mesothelioma, Ewing's tumor and other bone tumors (e.g.,        osteosarcoma, malignant fibrous histiocytoma), leiomyosarcoma,        rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast        cancer, ovarian cancer, prostate cancer, squamous cell        carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland        carcinoma, sebaceous gland carcinoma, papillary carcinoma,        papillary adenocarcinomas, cystadenocarcinoma, medullary        carcinoma, bronchogenic carcinoma, renal cell carcinoma,        hepatoma, bile duct carcinoma, choriocarcinoma, seminoma,        embryonal carcinoma, Wilms' tumor, cervical cancer, testicular        tumor, lung carcinoma, small cell lung carcinoma, bladder        carcinoma, epithelial carcinoma, brain/CNS tumors (e.g.,        astrocytoma, glioma, glioblastoma, childhood tumors, such as        atypical teratoid/rhabdoid tumor, germ cell tumor, embryonal        tumor, ependymoma) medulloblastoma, craniopharyngioma,        ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,        oligodendroglioma, meningioma, melanoma, neuroblastoma, and        retinoblastoma. Some of the most common solid tumors for which        the compositions and methods of the present disclosure would be        useful include: head-and-neck cancer, rectal adenocarcinoma,        glioma, medulloblastoma, urothelial carcinoma, pancreatic        adenocarcinoma, uterine (e.g., endometrial cancer, fallopian        tube cancer,) ovarian cancer, cervical cancer prostate        adenocarcinoma, non-small cell lung cancer (squamous and        adenocarcinoma), small cell lung cancer, melanoma, breast        carcinoma, ductal carcinoma in situ, renal cell carcinoma, and        hepatocellular carcinoma. adrenal tumors (e.g., adrenocortical        carcinoma), esophageal, eye (e.g., melanoma, retinoblastoma),        gallbladder, gastrointestinal, Wilms' tumor, heart, head and        neck, laryngeal and hypopharyngeal, oral (e.g., lip, mouth,        salivary gland), nasopharyngeal, neuroblastoma, peritoneal,        pituitary, Kaposi's sarcoma, small intestine, stomach,        testicular, thymus, thyroid, parathyroid, vaginal tumor and the        metastases of any of the foregoing.    -   “Metastasis” refers to the spread of cancer from its primary        site to neighboring tissues or distal locations in the body.        Cancer cells can break away from a primary tumor, penetrate into        lymphatic and blood vessels, circulate through the bloodstream,        and grow in in normal tissues elsewhere in the body. Metastasis        is a sequential process, contingent on tumor cells (or cancer        stem cells) breaking off from the primary tumor, traveling        through the bloodstream or lymphatics, and stopping at a distant        site. Once at another site, cancer cells re-penetrate through        the blood vessels or lymphatic walls, continue to multiply, and        eventually form a new tumor (metastatic tumor). In some        embodiments, this new tumor is referred to as a metastatic (or        secondary) tumor.    -   “Immune response” refers to the action of one or more of        lymphocytes, antigen presenting cells, phagocytic cells,        granulocytes, and soluble macromolecules produced by the above        cells or the liver (including antibodies, cytokines, and        complement) that results in selective damage to, destruction of,        or elimination from the human body of cancerous cells,        metastatic tumor cells, etc. An immune response may include a        cellular response, such as a T cell response that is an        alteration (modulation, e.g., significant enhancement,        stimulation, activation, impairment, or inhibition) of cellular        function that is a T cell function. A T cell response may        include generation, proliferation or expansion, or stimulation        of a particular type of T cell, or subset of T cells, for        example, effector CD4+, CD4⁺ helper, effector CD8+, CD8⁺        cytotoxic, or natural killer (NK) cells. Such T cell subsets may        be identified by detecting one or more cell receptors or cell        surface molecules (e.g., CD or cluster of differentiation        molecules). A T cell response may also include altered        expression (statistically significant increase or decrease) of a        cellular factor, such as a soluble mediator (e.g., a cytokine,        lymphokine, cytokine binding protein, or interleukin) that        influences the differentiation or proliferation of other cells.        For example, Type I interferon (IFN-α/β) is a critical regulator        of the innate immunity (52) (Huber et al. Immunology        132(4):466-474 (2011)). Animal and human studies have shown a        role for IFN-α/β in directly influencing the fate of both CD4⁺        and CD8⁺ T cells during the initial phases of antigen        recognition anti-tumor immune response. IFN Type I is induced in        response to activation of dendritic cells, in turn a sentinel of        the innate immune system.    -   “Tumor immunity” refers to one or more processes by which tumors        evade recognition and clearance by the immune system. Thus, as a        therapeutic concept, tumor immunity is “treated” when such        evasion is attenuated or eliminated, and the tumors are        recognized and attacked by the immune system (the latter being        termed herein “anti-tumor immunity”). An example of tumor        recognition is tumor binding, and examples of tumor attack are        tumor reduction (in number, size or both) and tumor clearance.    -   “T cell” refers to a thymus derived lymphocyte that participates        in a variety of cell-mediated adaptive immune reactions.    -   “Helper T cell” refers to a CD4⁺ T cell; helper T cells        recognize antigen bound to MHC Class II molecules. There are at        least two types of helper T cells, Th1 and Th2, which produce        different cytokines.    -   “Cytotoxic T cell” refers to a T cell that usually bears CD8        molecular markers on its surface (CD8+) and that functions in        cell-mediated immunity by destroying a target cell having a        specific antigenic molecule on its surface. Cytotoxic T cells        also release Granzyme, a serine protease that can enter target        cells via the perforin-formed pore and induce apoptosis (cell        death). Granzyme serves as a marker of cytotoxic phenotype.        Other names for cytotoxic T cell include CTL, cytolytic T cell,        cytolytic T lymphocyte, killer T cell, or killer T lymphocyte.        Targets of cytotoxic T cells may include virus-infected cells,        cells infected with bacterial or protozoal parasites, or cancer        cells. Most cytotoxic T cells have the protein CD8 present on        their cell surfaces. CD8 is attracted to portions of the Class I        MHC molecule. Typically, a cytotoxic T cell is a CD8+ cell.    -   “Tumor-infiltrating leukocytes” refers to white blood cells of a        subject afflicted with a cancer (such as melanoma), that are        resident in or otherwise have left the circulation (blood or        lymphatic fluid) and have migrated into a tumor.    -   “Immune checkpoint inhibitor” or “immune checkpoint blocking        agent” refers to molecules that completely or partially reduce,        inhibit, interfere with or modulate the activity of one or more        checkpoint proteins. Checkpoint proteins regulate T-cell        activation or function. Checkpoint proteins include, but are not        limited to CD28 receptor family members, CTLA-4 and its ligands        CD80 and CD86; PD-1 and its ligands PDL1 and PDL2; LAGS, B7-H3,        B7-H4, TIM3, ICOS, and BTLA (53).    -   “Parenteral” when used in the context of administration of a        therapeutic substance includes any route of administration other        than administration through the alimentary tract. Particularly        relevant for the methods disclosed herein are intravenous        (including for example through the hepatic portal vein),        intratumoral or intrathecal administration.    -   “Antibody” refers to an immunoglobulin molecule which        specifically binds to an antigen or to an antigen-binding        fragment of such a molecule. Thus, antibodies can be intact        immunoglobulins derived from natural sources or from recombinant        sources and can be immunoreactive (antigen-binding) fragments or        portions of intact immunoglobulins. The antibodies may exist in        a variety of forms including, for example, polyclonal        antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well        as single chain antibodies (scFv) humanized antibodies, chimeric        antibodies, human recombinant antibodies and bi- and        tri-specific antibodies.    -   “Oncolytic virus” refers to a virus that preferentially infects        cancer cells, replicates in such cells, and induces lysis of the        cancer cells through its replication process. Nonlimiting        examples of naturally occurring oncolytic viruses include        vesicular stomatitis virus, reovirus, as well as viruses        engineered to be oncoselective such as adenovirus, Newcastle        disease virus and herpes simplex virus (See, e.g.,        Nemunaitis, J. Invest New Drugs. 17(4):375-86 (1999); Kim, D H        et al. Nat Rev Cancer. 9(1):64-71(2009); Kim et al. Nat. Med.        7:781 (2001); Coffey et al. Science 282:1332 (1998)) (8, 54-56).        Vaccinia virus infects many types of cells but replicates        preferentially in tumor cells due to the fact that tumor cells        have a metabolism that favors replication, exhibit activation of        certain pathways that also favor replication and create an        environment that evades the innate immune system, which also        favors viral replication. In the context of the present        disclosure, MVA and MVAΔE3L do not fit the definition of        oncolytic viruses as they do not produce an antitumor effect        primarily by replicating inside tumor cells and causing        apoptosis. (Nor do they fit the classic definition of vaccines        as these viruses do not express tumor antigens. It can be said        however, that they act as immunostimulatory molecules, akin to        adjuvants, as they serve to enhance the host's immune response        against the tumor.)    -   “MVA” means “modified vaccinia Ankara” and refers to a highly        attenuated strain of vaccinia derived from the Ankara strain and        developed for use as a vaccine and vaccine adjuvant. The        original MVA was isolated from the wild-type Ankara strain by        successive passage through chicken embryonic cells, Treated        thus, it lost about 15% of the genome of wild-type vaccinia        including its ability to replicate efficiently in primate        (including human) cells. (57) (Mayr et al., Zentralbl Bakteriol        B167, 375-390 (1978)). The smallpox vaccination strain MVA:        marker, genetic structure, experience gained with the parenteral        vaccination and behavior in organisms with a debilitated defense        mechanism. MVA is considered an appropriate candidate for        development as a recombinant vector for gene or vaccination        delivery against infectious diseases or tumors. (58) (Verheust        et al., Vaccine 30(16), 2623-2632 (2012)). MVA has a genome of        178 kb in length and a sequence first disclosed in (59) (Antoine        et al., Virol. 244(2): 365-396 (1998)). Sequences are also        disclosed in Genbank U94848.1. Clinical grade MVA is        commercially and publicly available from Bavarian Nordic A/S        Kvistgaard, Denmark. Additionally, MVA is available from ATCC,        Rockville, Md. and from CMCN (Institut Pasteur Collection        Nationale des Microorganismes) Paris, France.    -   “MVAΔE3L” means a deletion mutant of MVA which lacks a        functional E3L gene and is infective but non replicative and it        is further impaired in its ability to evade the host's immune        system. It can be used as a vaccine vector. This mutant MVA E3L        knockout and its preparation have been described for example in        U.S. Pat. No. 7,049,145.    -   “Subject” means any animal (mammalian, human or other) patient        that can be afflicted with cancer and when thus afflicted is in        need of treatment.    -   “Therapeutically effective amount” or “effective amount” refers        to a sufficient amount of an agent when administered at one or        more dosages and for a period of time sufficient to provide a        desired biological result in alleviating, curing or palliating a        disease. In the present disclosure, an effective amount        respectively of the MVA or MVAΔE3L is an amount that        (administered for a suitable period of time and at a suitable        frequency) reduces the number of cancer cells; or reduces the        tumor size or eradicates the tumor; or inhibits (i.e., slows        down or stops) cancer cell infiltration into peripheral organs;        inhibits (i.e., slows down or stops) metastatic growth; inhibits        (stabilizes or arrests) tumor growth; allows for treatment of        the tumor, and/or induces an immune response against the tumor.        An appropriate therapeutic amount in any individual case may be        determined by one of ordinary skill in the art using routine        experimentation in light of the present disclosure. Such        determination will begin with amounts found effective in vitro        and amounts found effective in animals. The therapeutically        effective amount will be initially determined based on the        concentration or concentrations found to confer a benefit to        cells in culture. Effective amounts can be extrapolated from        data within the cell culture and can be adjusted up or down        based on factors such as detailed herein. An example of an        effective amount range is from 10⁵ viral particles to about 10¹²        viral particles per administration.

With particular reference to the viral-based immunostimulatory agentsdisclosed herein, “therapeutically effective amount” or “effectiveamount” refers to an amount of a composition comprising MVA or MVAΔE3Lsufficient to reduce, inhibit, or abrogate tumor cell growth, therebyreducing or eliminating the tumor, or sufficient to inhibit, reduce orabrogate metastatic spread either in vitro, ex vivo or in a subject orto elicit an immune response against the tumor that will eventuallyresult in one or more of metastatic spread reduction, inhibition and/orabrogation as the case may be. The reduction, inhibition, or eradicationof tumor cell growth may be the result of necrosis, apoptosis, or animmune response or a combination of two or more of the foregoing(however, the precipitation of apoptosis for example may not be due tothe same factors as observed with oncolytic viruses). The amount that istherapeutically effective may vary depending on such factors as theparticular MVA used in the composition, the age and condition of thesubject being treated, the extent of tumor formation, the presence orabsence of other therapeutic modalities, and the like. Similarly, thedosage of the composition to be administered and the frequency of itsadministration will depend on a variety of factors, such as the potencyof the active ingredient, the duration of its activity onceadministered, the route of administration, the size, age, sex andphysical condition of the subject, the risk of adverse reactions and thejudgment of the medical practitioner. The compositions are administeredin a variety of dosage forms, such as injectable solutions.

With particular reference to combination therapy with an immunecheckpoint inhibitor, “therapeutically effective amount” for an immunecheckpoint blocking agent” shall mean an amount of an immune checkpointblocking agent sufficient to reverse or reduce immune suppression in thetumor microenvironment and to activate or enhance host immunity in thesubject being treated. There are several immune checkpoint blockingagents approved, in clinical trials or still otherwise under developmentincluding inhibitory antibodies against CD28 inhibitor such as CTLA-4(cytotoxic T lymphocyte antigen 4) (e.g., ipilimumab), anti-PD-1(programmed Death 1) inhibitory antibodies (e.g., nivolumab,pembrolizumab, pidilizumab, lambrolizumab), and anti-PD-L1 (Programmeddeath ligand 1) inhibitory antibodies (MPDL3280A, BMS-936559, MEDI4736,MSB 00107180), as well as inhibitory antibodies against LAG-3(lymphocyte activation gene 3), TIM3 (T cell Immunoglobulin andMucin-3), B7-H3, and TIGIT (T-cell immunoreceptor with Ig and ITIMdomains). Dosage ranges of the foregoing are known in or readily withinthe skill in the art as several dosing clinical trials have beencompleted, making extrapolation to other agents possible.

Preferably, the tumor expresses the particular checkpoint but this isnot strictly necessary as immune checkpoint blocking agents block moregenerally immune suppressive mechanisms within the tumors, elicited bytumor cells, stromal cells, and tumor-infiltrating immune cells.

For example, the CTLA4 inhibitor ipilimumab, when administered asadjuvant therapy after surgery in melanoma is administered at 1-2 mg/mLover 90 minutes for a total infusion amount of 3 mg/kg every three weeksfor a total of 4 doses. This therapy is often accompanied by severe evenlife-threatening immune-mediated adverse reactions, which limits thetolerated dose as well as the cumulative amount that can beadministered. It is anticipated that it will be possible to reduce thedose and/or cumulative amount of ipilimumab when it is administeredconjointly with MVA or MVAΔE3L. In particular, in light of theexperimental results set forth below, it is anticipated that it will befurther possible to reduce the CTLA4 inhibitor's dose if it isadministered directly to the tumor simultaneously or sequentially withone or both the foregoing MVA viruses. Accordingly, the amounts providedabove for ipilimumab will be a starting point for determining theparticular dosage and cumulative amount to be given to a patient inconjoint administration but dosing studies will be required to determineoptimum amounts.

Pembrolizumab is prescribed for administration as adjuvant therapy inmelanoma diluted to 25 mg/mL. It is administered at a dosage of 2 mg/kgover 30 minutes every three weeks. Again, this would be a starting pointfor determining dosage and administration in the conjoint administrationwith MVA or MVAΔE3L.

Nivolumab is prescribed for administration at 3 mg/kg as an intravenousinfusion over 60 minutes every two weeks, providing a similar startingpoint in determining dosage and administration of this checkpointinhibitor conjointly with MVA or MVAΔE3L.

Immune stimulating agents such as agonist antibodies have also beenexplored as immunotherapy for cancers. For example, anti-ICOS antibodybinds to the extracellular domain of ICOS leading to the activation ofICOS signaling and T cell activation. Anti-OX40 antibody can bind toOX40 and potentiate T cell receptor signaling leading to T cellactivation, proliferation and survival. Other examples include agonistantibodies against 4-1BB (CD137), GITR. All of these agents are atvarious stages of clinical trials.

The immune stimulating agonist antibodies can be used systemically incombination with intratumoral injection of MVA or MVAΔE3L.Alternatively, the immune stimulating agonist antibodies can be usedconjointly with MVA or MVAΔE3L via intratumorally delivery eithersimultaneously or sequentially.

-   -   “Pharmaceutically acceptable carrier and/or diluent” or        “pharmaceutically acceptable excipient” includes without        limitation any and all solvents, dispersion media, coatings,        antibacterial and antifungal agents, isotonic and absorption        delaying agents and the like. The use of such media and agents        for biologically active substances is well known in the art.        Further details of excipients are provided below. Supplementary        active ingredients, such as antimicrobials, for example        antifungal agents, can also be incorporated into the        compositions.    -   “Delivering” used in connection with depositing the MVA or        MVAΔE3L of the present disclosure in the tumor microenvironment        whether this is done by local administration to the tumor or by        for example intravenous route. The term focuses on MVA or        MVAΔE3L that reaches the tumor itself.    -   “Conjoint administration” herein refers to administration of a        second therapeutic modality in combination with MVA or MVAΔE3L        for example an immune checkpoint blocking agent administered and        in close temporal proximity with MVA or MVAΔE3L. For example, a        PD-1/PDL-1 inhibitor and/or a CTLA4 inhibitor (in more specific        embodiments, an antibody) can be administered simultaneously        with MVA or MVAΔE3L (by intravenous or intratumoral injection        when the MVA or MVAΔE3L is administered intratumorally or        systemically as stated above) or before or after the MVA or        MVAΔE3L administration. If the MVA or MVAΔE3L administration and        the immune checkpoint blocking agent are administered 1-7 days        apart or even up to three weeks apart, this would be within        “close temporal proximity” as stated herein.

In one embodiment, the present disclosure relates to a method foreliciting an antitumor immune response in subjects afflicted with tumorscomprising delivering to the tumor an effective amount of MVA orMVAΔE3L. Stimulation of the immune system may be manifest by one or moreof the following immunological effects:

-   -   an increase in at least one of effector CD8⁺ T cells and        effector CD4+ T cells within the tumor and/or in tumor-draining        lymph nodes;    -   induction of maturation of dendritic cells infiltrating said        tumor through induction of type I IFN;    -   induction of effector CD4⁺ T cells in the subject recognizing        tumor cells within the tumor and/or in tumor draining lymph        nodes;    -   reduction of immune suppressive (regulatory) CD4⁺ T cells within        the tumor;    -   induction of cells of the tumor to produce one or more of Type I        IFN or other inflammatory cytokines or chemokines;    -   reduction of immune suppressive tumor-associated macrophages        within the tumor.

The foregoing one or more immunological effects may serve as earlyindicators of response of the subject to the treatment and may serve asmonitors of the continued effectiveness of same. Observation of theseeffects illustrates that the manner in which the present viruses treattumor is different from that of vaccine vectors harboring tumor antigens(which are not delivered intratumorally but by intramuscular,subcutaneous or, rarely, intravenous route) and also different from thatof oncolytic viruses (which cause cytopathy primarily due to viralreplication in tumor cells). If apoptosis results pursuant to thepresent treatment, it is not due to the same mechanism as apoptosis thatresults or may result from these different modes of action.

The present inventors have explored the mechanism of the immune responseand concluded that it is initiated by the cytosolic DNA-sensing pathwaymediated by cGAS/STING which mediates production of Type 1 IFN. Furtherinsights into the mechanism and the immune cells that are recruited areprovided in the Examples. The conclusions presented therein are notconfined to the specific experimental milieu where these mechanisms arebeing elucidated.

In one embodiment, the present disclosure provides a method of treatinga subject diagnosed with a solid tumor comprising delivering to thetumor a therapeutic effective amount of the MVA or MVAΔE3L.

In one embodiment, the present disclosure provides a method for inducinganti-tumor immunity in a subject diagnosed with cancer comprisingadministering to the subject a therapeutically effective amount of MVAor MVAΔE3L. The methods of the present disclosure include induction ofanti-tumor immunity that can reduce the size of the tumor, eradicate thetumor, inhibit growth of the tumor, inhibit metastasis or reducemetastatic growth of the tumor or eradicate metastatic growth of thetumor, induce apoptosis of tumor cells or prolong survival of thesubject (compared to untreated or conventionally treated subjects).

In another embodiment, the present disclosure provides a method forenhancing, stimulating, or eliciting, in a subject diagnosed with asolid malignant tumor, an anti-tumor immune response that may include aninnate immune response and/or an adaptive immune response such as a Tcell response by exposing the tumor to MVA or MVAΔE3L in atherapeutically effective amount.

In specific embodiments, the present disclosure provides methods ofeliciting an immune response that mediates adaptive immune responsesboth in terms of T-cell cytotoxicity directed against tumor cells and interms of eliciting effector T cells also directed against tumor cells.The methods comprise administering to a subject afflicted with a solidtumor intratumorally or (as the present inventors anticipate)intravenously a composition comprising MVA or MVAΔE3L whereinadministration of said composition results in a tumor-specific immuneresponse against the tumor and, eventually, in reduction, inhibition oreradication of tumor growth, inhibition of metastatic growth, apoptosisof tumor cells and/or prolongation of the subject's survival. Indeed thepresent inventors have shown that cancer cells are being killed and thatthe immune response can migrate to remote locations, as would be thecase with metastases.

In some embodiments, the present disclosure provides methods ofeliciting an immune response that mediates adaptive immune responsesboth in terms of T-cell cytotoxicity directed against tumor cells and interms of eliciting effector T cells also directed against tumor cells.The methods comprise administering to a subject parenterally acomposition comprising MVA or MVAΔE3L wherein administration of saidcomposition results in a tumor-specific immune response against thetumor and, eventually, in reduction, inhibition or eradication of tumorgrowth and/or in inhibition reduction or elimination of metastaticgrowth, apoptosis of tumor cells and/or prolongation of survival of thetreated subject compared to conventional therapy or no treatment. Forintraperitoneal metastases, the virus can be injected intraperitoneally.

Indeed the present inventors have shown that cancer cells are beingkilled and that the immune response can migrate to remote locations, aswould be the case with metastases, and still exert an anti-tumor effect.

Because MVA and MVAΔE3L are substantially not replication competent inmost mammalian cells, it does not exert its effect on the immune systemthe same way as replication competent vaccines or vectors. Thus, whileit is believed that stimulation of the immune system is a barrier toefficacy for oncolysis (8) (Kirn et al., Nat Rev Cancer. (1), 64-71(2009)), MVA and MVAΔE3L is able to harness the innate immune system tostimulate adaptive immunity, both in terms of cytotoxicity and morebroadly in terms of effector T cell activation against the tumor.

The present disclosure thus provides a method for treating a solidmalignant tumor, comprising delivering to a tumor of the subject anamount of MVA or MVAΔE3L effective to induce an immune response againstthe tumor in a subject diagnosed with solid tumor.

The present disclosure also provides a method for generating antitumorsystemic immunity in a subject afflicted with a solid malignant tumor,comprising delivering to a tumor of the subject an amount of MVA orMVAΔE3L effective to bring about one or both of rejection ofnon-injected tumors in said subject and inhibition of tumor metastasis(which the present inventors test by tumor rechallenge).

As is shown herein, MVA induces type I IFN induction in conventionaldendritic cells (cDCs) via a cytosolic DNA-sensing pathway mediated bycGAS/STING. Intravenous delivery of MVA in C57B/6 mice induced type IIFN in wild-type mice, but not in mice lacking STING or IRF3. It is alsoshown that MVAΔE3L induces higher levels of type I IFN gene expressionand phosphorylation of IRF3 than MVA in cDCs. MVAΔE3L is detected byboth the cytosolic DNA-sensing pathway mediated by cGAS/STING, and thedsRNA-sensing pathway mediated by MDA5/MAVS. In addition, MVAΔE3Linfection of B16 melanoma cells and MC38 colon adenocarcinoma cellsinduces type I IFN and proinflammatory cytokines and chemokines, as wellas activation of phosphorylation of IRF3. Both MVA and MVAΔE3L induceapoptosis in B16 and MC38 cells as demonstrated by the cleavage of PARPand Caspase-3. According to the present disclosure MVA or MVAΔE3L virusis used as direct anti-cancer therapy. Intratumoral injection of MVA orMVAΔE3L in a murine B16 melanoma model leads to apoptosis, prolongedsurvival and tumor eradication, as well as the generation of systemicanti-tumor immunity.

Based on current literature, and without wishing to be bound by theory,the following mechanisms are believed to contribute to anti-tumoreffects of MVA and MVAΔE3L: (i) induction of type I IFN responses inimmune cells including conventional dendritic cells and macrophages;(ii) induction of type I IFN and proinflammatory cytokines andchemokines in cancer cells; (iii) induction of apoptosis in cancercells; and (iv) alteration of tumor immune suppressive environment to animmune activating one.

Modified Vaccinia Ankara (MVA)

Modified Vaccinia Ankara (MVA) virus is a member of the generaOrthopoxvirus in the family of Poxviridae. MVA was generated byapproximately 570 serial passages on chicken embryo fibroblasts (CEF) ofthe Ankara strain of vaccinia virus (CVA) (60) (Mayr et al., Infection3, 6-14 (1975)). As a consequence of these long-term passages, theresulting MVA virus contains extensive genome deletions and is highlyhost cell restricted to avian cells (61) (Meyer et al., J. Gen. Virol.72, 1031-1038 (1991)). It was shown in a variety of animal models thatthe resulting MVA is significantly avirulent (57) (Mayr et al., Dev.Biol. Stand. 41, 225-34 (1978)).

The safety and immunogenicity of MVA has been extensively tested anddocumented in clinical trials, particularly against the human smallpoxdisease. These studies included over 120,000 individuals and havedemonstrated excellent efficacy and safety in humans. Moreover, comparedto other vaccinia based vaccines, MVA has weakened virulence(infectiousness) while it triggers a good specific immune response.Thus, MVA has been established as a safe vaccine vector, with theability to induce a specific immune response.

Due to the above mentioned characteristics, MVA became an attractivecandidate for the development of engineered MVA vectors, used forrecombinant gene expression and vaccines. As a vaccine vector, MVA hasbeen investigated against numerous pathological conditions, includingHIV, tuberculosis and malaria, as well as cancer (20, 21) (Sutter etal., Curr Drug Targets Infect Disord 3: 263-271(2003); Gomez et al.,Curr Gene Ther 8: 97-120 (2008)).

It has been demonstrated that MVA infection of human monocyte-deriveddendritic cells (DC) causes DC activation, characterized by theupregulation of co-stimulatory molecules and secretion ofproinflammatory cytokines (18) (Drillien et al., J Gen Virol 85:2167-2175 (2004)). In this respect, MVA differs from standard wild typeVaccinia virus (WT-VAC), which fails to activate DCs. Dendritic cellscan be classified into two main subtypes: conventional dendritic cells(cDCs) and plasmacytoid dendritic cells (pDCs). The former, especiallythe CD103+/CD8α⁺ subtype, are particularly adapted to cross-presentingantigens to T cells; the latter are strong producers of Type I IFN.

Viral infection of human cells results in activation of an innate immuneresponse (the first line of defense) mediated by type I interferons,notably interferon-alpha (a). This normally leads to activation of animmunological “cascade,” with recruitment and proliferation of activatedT cells (both CTL and helper) and eventually with antibody production.However viruses express factors that dampen immune responses of thehost. MVA is a better immunogen than WT-VAC and replicates poorly inmammalian cells. (See, e.g., Brandler et al., J. Virol. 84, 5314-5328(2010)) (62).

However, MVA is not entirely nonreplicative and as the present inventorsshow contains some residual immunosuppressive activity. Nevertheless, asshown herein MVA significantly prolonged survival of treated subjects.An implication of these findings is that by injecting a tumor with orsystemically delivering MVA (or MVAΔE3L) it is possible to enhance ahost's innate and adaptive immune responses and thereby overcome thetumor's ability to evade immune responses and to restore the ability ofthe host to mount an immune response against the tumor whether theresponse is native or induced or enhanced by another immunotherapeuticagent, such as a checkpoint inhibitor.

Modified Vaccinia Ankara with Deletion of E3 (MVAΔE3L)

The antitumor effects of MVA described in the immediately precedingsection are also observed with MVAΔE3L. The latter is lessimmunosuppressive than MVA and even less replicative in most mammaliancells, and from that point of view preferred. In addition, the effectsof MVAΔE3L have generally been qualitatively better than those with MVAas seen in the experiments described herein.

Immune Response

In addition to induction of the immune response by up-regulation ofparticular immune system activities (such as antibody and/or cytokineproduction, or activation of cell mediated immunity), immune responsesmay also include suppression, attenuation, or any other downregulationof detectable immunity, so as to reestablish homeostasis and preventexcessive damage to the host's own organs and tissues. In someembodiments, an immune response that is induced according to the methodsof the present disclosure generates effector CD8⁺ (antitumor cytotoxicCD8⁺) T cells or activated T helper cells or both that can bring aboutdirectly or indirectly the death, or loss of the ability to propagate,of a tumor cell.

Induction of an immune response by the methods of the present disclosuremay be determined by detecting any of a variety of well-knownimmunological parameters (63, 64) (Takaoka et al., Cancer Sci. 94:405-11(2003); Nagorsen et al., Crit. Rev. Immunol. 22:449-62 (2002)).Induction of an immune response may therefore be established by any of anumber of well-known assays, including immunological assays, Such assaysinclude, but need not be limited to, in vivo, ex vivo, or in vitrodetermination of soluble immunoglobulins or antibodies; solublemediators such as cytokines, chemokines, hormones, growth factors andthe like as well as other soluble small peptide, carbohydrate,nucleotide and/or lipid mediators; cellular activation state changes asdetermined by altered functional or structural properties of cells ofthe immune system, for example cell proliferation, altered motility,altered intracellular cation gradient or concentration (such ascalcium); phosphorylation or dephosphorylation of cellular polypeptides;induction of specialized activities such as specific gene expression orcytolytic behavior; cellular differentiation by cells of the immunesystem, including altered surface antigen expression profiles, or theonset of apoptosis (programmed cell death); or any other criterion bywhich the presence of an immune response may be detected. For example,cell surface markers that distinguish immune cell types may be detectedby specific antibodies that bind to CD4+, CD8+, or NK cells. Othermarkers and cellular components that can be detected include but are notlimited to interferon γ (IFN-γ), tumor necrosis factor (TNF), IFN-α,IFN-β, IL-6, and CCL5. Common methods for detecting the immune responseinclude, but are not limited to flow cytometry, ELISA,immunohistochemistry. Procedures for performing these and similar assaysare widely known and may be found, for example in Letkovits (ImmunologyMethods Manual: The Comprehensive Sourcebook of Techniques, CurrentProtocols in Immunology, 1998).

Pharmaceutical Compositions and Preparations

Pharmaceutical compositions comprising MVA or MVAΔE3L may contain acarrier or diluent, which can be a solvent or dispersion mediumcontaining, for example, water, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be effectedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin. In general, excipients suitable for injectable preparations canbe included as apparent to those skilled in the art.

Pharmaceutical compositions and preparations comprising MVA or MVAΔE3Lmay be manufactured by means of conventional mixing, dissolving,granulating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical viral compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries that facilitateformulating virus preparations suitable for in vitro, in vivo, or exvivo use. The compositions can be combined with one or more additionalbiologically active agents (for example parallel administration ofGM-CSF) and may be formulated with a pharmaceutically acceptablecarrier, diluent or excipient to generate pharmaceutical (includingbiologic) or veterinary compositions of the instant disclosure suitablefor parenteral or intra-tumoral administration.

Many types of formulation are possible as is appreciated by thoseskilled in the art. The particular type chosen is dependent upon theroute of administration chosen, as is well-recognized in the art. Forexample, systemic formulations will generally be designed foradministration by injection, e.g., intravenous, as well as thosedesigned for intratumoral delivery. Preferably, the systemic orintratumoral formulation is sterile.

Sterile injectable solutions are prepared by incorporating MVA orMVAΔE3L in the required amount of the appropriate solvent with variousother ingredients enumerated herein, as required, followed by suitablesterilization means. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying techniques,which yield a powder of the virus plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

In some embodiments, the MVA and MVAΔE3L compositions of the presentdisclosure may be formulated in aqueous solutions, or in physiologicallycompatible solutions or buffers such as Hanks's solution, Ringer'ssolution, mannitol solutions or physiological saline buffer. In certainembodiments, any of the MVA and MVAΔE3L compositions may containformulator agents, such as suspending, stabilizing, penetrating ordispersing agents, buffers, lyoprotectants or preservatives such aspolyethylene glycol, polysorbate 80, 1-dodecylhexahydro-2H-azepin-2-one(laurocapran), oleic acid, sodium citrate, Tris HCl, dextrose, propyleneglycol, mannitol, polysorbate polyethylenesorbitan monolaurate(Tween®-20), isopropyl myristate, benzyl alcohol, isopropyl alcohol,ethanol sucrose, trehalose and other such generally known in the art maybe used in any of the compositions of the instant disclosure. (Pramanicket al., Pharma Times 45(3), 65-76 (2013)) (65).

The biologic or pharmaceutical compositions of the present disclosurecan be formulated to allow the virus contained therein to be availableto infect tumor cells upon administration of the composition to asubject. The level of virus in serum, tumors, and if desired othertissues after administration can be monitored by variouswell-established techniques, such as antibody-based assays (e.g., ELISA,immunohistochemistry, etc.).

Dosage of MVA and MVAΔE3L

In general, the subject is administered a dosage of MVA and MVAΔE3L inthe range of about 10⁵ to about 10¹⁰ plaque forming units (pfu),although a lower or higher dose may be administered. In a preferredembodiment, dosage is about 10⁶-10⁹ pfu. The equivalence of pfu to virusparticles can differ according to the specific pfu titration methodused. Generally, pfu is equal to about 5 to 100 virus particles. Atherapeutically effective amount of MVA or MVAΔE3L can be administeredin one or more divided doses for a prescribed period of time and at aprescribed frequency of administration.

For example, as is apparent to those skilled in the art, atherapeutically effective amount of MVA or MVAΔE3L in accordance withthe present disclosure may vary according to factors such as the diseasestate, age, sex, weight, and general condition of the subject, and theability of MVA or MVAΔE3L to elicit a desired immunological response inthe particular subject (the subject's response to therapy). Indelivering MVA or MVAΔE3L to a subject, the dosage will also varydepending upon such factors as the general medical condition, previousmedical history, disease progression, tumor burden and the like.

In some embodiments, it may be advantageous to formulate compositions ofpresent disclosure in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required pharmaceutically or veterinary acceptablecarrier.

Administration and Therapeutic Regimen of MVA and MVAΔE3L

Administration of MVA and MVAΔE3L can be achieved using more than oneroute, including parenteral, for example intratumoral or intravenous,administration. In one embodiment, MVA or MVAΔE3L is administereddirectly into the tumor, e.g. by intratumoral injection, where a directlocal reaction is desired. Additionally, administration routes of MVAand MVAΔE3L can vary, e.g., first administration using an intratumoralinjection, and subsequent administration via an intravenous injection,or any combination thereof. A therapeutically effective amount of MVA orMVAΔE3L injection can be administered for a prescribed period of timeand at a prescribed frequency of administration. In certain embodiments,MVA and MVAΔE3L can be used in conjunction with other therapeutictreatments. For example, MVA and MVAΔE3L can be administered in aneoadjuvant (preoperative) or adjuvant (postoperative) setting forsubjects inflicted with bulky primary tumors. It is anticipated thatsuch optimized therapeutic regimen will induce an immune responseagainst the tumor, and reduce the tumor burden in a subject before orafter primary therapy, such as surgery. Furthermore, MVA or MVAΔE3L canbe administered in conjunction with other therapeutic treatments such aschemotherapy or radiation.

In certain embodiments, the MVA or MVAΔE3L virus is administered atleast once weekly or monthly but can be administered more often ifneeded, such as two times weekly for several weeks, months, years oreven indefinitely as long as benefits persist. More frequentadministrations are contemplated if tolerated and if they result insustained or increased benefits. Benefits of the present methods includebut are not limited to the following: reduction of the number of cancercells, reduction of the tumor size, eradication of tumor, inhibition ofcancer cell infiltration into peripheral organs, inhibition orstabilization or eradication of metastatic growth, inhibition orstabilization of tumor growth, and stabilization or improvement ofquality of life. Furthermore, the benefits may include induction of animmune response against the tumor, activation of effector CD4 T cells,an increase of effector CD8⁺ T cells, or reduction of regulatory CD4⁺cells. For example, in the context of melanoma or, a benefit may be alack of recurrences or metastasis within one, two, three, four, five ormore years of the initial diagnosis of melanoma. Similar assessments canbe made for colon cancer and other solid tumors.

In certain other embodiments, the tumor mass or tumor cells are treatedwith MVA or MVAΔE3L in vivo, ex vivo, or in vitro.

EXAMPLES Materials and Methods Viruses and Cell Lines

MVA and MVAΔE3L viruses were kindly provided by Gerd Sutter (Universityof Munich), and propagated in BHK-21 (baby hamster kidney cell, ATCCCCL-10) cells. MVA is commercially and/or publicly available. The methodof generation of MVAΔE3L Viruses was described (28) (Hornemann et al., JVirol 77, 8394-8407 (2003)). The viruses were purified through a 36%sucrose cushion. BHK-21 were cultured in Eagle's Minimal EssentialMedium (Eagle's MEM, can be purchased from Life Technologies,Cat#11095-080) containing 10% FBS, 0.1 mM nonessential amino acids(NEAA), and 50 mg/ml gentamycin. The murine melanoma cell line B16-F10was originally obtained from I. Fidler (MD Anderson Cancer Center).B16-F10 cells were maintained in RPMI 1640 medium supplemented with 10%FBS, 100 Units/ml penicillin, 100 μg/ml streptomycin, 0.1 mM NEAA, 2 mML-glutamine, 1 mM sodium pyruvate, and 10 mM HEPES buffer. The MC38colon adenocarcinoma cancer cells were maintained in Dulbecco's modifiedEagle medium (DMEM, Invitrogen). All cells were grown at 37° C. in a 5%CO₂ incubator.

Cells and cell lines used herein are commercially or publicly availableunless otherwise indicated.

Mice

Female C57BL/6J mice between 6 and 10 weeks of age were purchased fromthe Jackson Laboratory and were used for the preparation of bonemarrow-derived dendritic cells and as control mice for in vivoexperiments. These mice were maintained in the animal facility at theSloan Kettering Institute. All procedures were performed in strictaccordance with the recommendations in the Guide for the Care and Use ofLaboratory Animals of the National Institute of Health. The protocol wasapproved by the Committee on the Ethics of Animal Experiments ofSloan-Kettering Cancer Institute. cGAS^(−/−), IRF3^(−/−), IRF7^(−/−),MAVS^(−/−), MDA5^(−/−), and STING^(Gt/Gt) mice were generated in thelaboratories of Drs. Zhijian Chen (University of Texas SouthwesternMedical Center; cGAS^(−/−) and MAVS^(−/−)), Tadatsugu Taniguchi(University of Tokyo; IRF3^(−/−) and IRF7^(−/−)), Marco Colonna(Washington University, MDA5^(−/−)), and Russell Vance (University ofCalifornia, Berkeley; STING^(Gt/Gt)).

Commercial sources for these or comparable animals are as follows:

Mice Source Commercial Source cGAS^(−/−) Zhijian Jackson Stock# 026554Chen MAVS^(−/−) Zhijian Jackson stock# 008634 Chen MDA5^(−/−) MarcoJackson stock# 015812 Colonna STING^(Gt/Gt) Russell Jackson stock#017537 Vance IRF3^(−/−) T. Taniguchi lab Taniguchihttp://www2.brc.riken.jp/lab/animal/detail.php?reg_no=RBRC00858IRF7^(−/−) T. Taniguchi lab Taniguchihttps://www2.brc.riken.jp/lab/animal/detail.php?brc no=RBRC01420

Generation of Bone Marrow-Derived Dendritic Cells

The bone marrow cells from the tibia and femur of mice were collected byfirst removing muscles from the bones, and then flushing the cells outusing 0.5 cc U-100 insulin syringes (Becton Dickinson) with RPMI with10% FCS. After centrifugation, cells were re-suspended in ACK LysingBuffer (Lonza) for red blood cells lysis by incubating the cells on icefor 1-3 min. Cells were then collected, re-suspended in fresh medium,and filtered through a 40-μm cell strainer (BD Biosciences). The numberof cells was counted. For the generation of GM-CSF-BMDCs, the bonemarrow cells (5 million cells in each 15 cm cell culture dish) werecultured in CM in the presence of GM-CSF (30 ng/ml, produced by theMonoclonal Antibody Core facility at the Sloan Kettering Institute) for10-12 days. CM is RPMI 1640 medium supplemented with 10% fetal bovineserum (FBS), 100 Units/ml penicillin, 100 μg/ml streptomycin, 0.1 mMessential and nonessential amino acids, 2 mM L-glutamine, 1 mM sodiumpyruvate, and 10 mM HEPES buffer. Cells were fed every 2 days byreplacing 50% of the old medium with fresh medium and re-plated every3-4 days to remove adherent cells. Only non-adherent cells were used forexperiments.

RNA Isolation and Real-Time PCR

RNA was extracted from whole-cell lystates with an RNeasy Mini kit(Qiagen) and was reverse transcribed with a First Strand cDNA synthesiskit (Fermentas). Quantitative real-time PCR was performed in triplicatewith SYBR Green PCR Mater Mix (Life Technologies) and Applied Biosystems7500 Real-time PCR Instrument (Life Technologies) using gene-specificprimers. Relative expression was normalized to the levels ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH).

The following primers were used for real-time PCR:

IFNA4 forward: 5′-CCTGTGTGATGCAGGAACC-3′, IFNA4 reverse:5′-TCACCTCCCAGGCACAGA-3′; IFNB forward: 5′-TGGAGATGACGGAGAAGATG-3′,IFNB reverse: 5′-TTGGATGGCAAAGGCAGT-3′; CCL5 forward:5′-GCCCACGTCAAGGAGTATTTCTA-3′, CCL5 reverse: 5′-ACACACTTGGCGGTTCCTTC-3′;IL-6 forward: 5′-AGGCATAACGCACTAGGTTT-3′, IL-6 reverse:5′-AGCTGGAGTCACAGAAGGAG-3′; CXCL10 forward: 5′ ATTCTTTAAGGGCTGGTCTGA 3′CXCL10 reverse: 5′ CACCTCCACATAGCTTACAGT 3′ TNF forward: 5′GTCAGGTTGCCTCTGTCTCA 3′ TNF reverse: 5′ TCAGGGAAGAGTCTGGAAAG 3′GAPDH forward: 5′-ATCAAGAAGGTGGTGAAGCA-3′, GAPDH reverse:5′-AGACAACCTGGTCCTCAGTGT-3′.

Relative expression was normalized to the levels ofglyceraldehyde-3-phosphate dehydrogenase (GADPH).

Cytokine Assays

Cells were infected with various viruses at a MOI of 10 for 1 h or mockinfected. The inoculum was removed and the cells were washed with PBStwice and incubated with fresh medium. Supernatants were collected atvarious times post infection. Cytokine levels were measured by usingenzyme-linked immunosorbent essay (ELISA) kits for IFN-α/β (PBLBiomedical Laboratories), IL-6, CCL4, and CCL5 (R & D systems).

Western Blot Analysis

BMDCs, B16-F10, or MC38 cells (1×10⁶) were infected with MVA at a MOI(multiplicity of infection) of 10 or an equivalent amount of MVA orMVAΔE3L. At various times post-infection, the medium was removed andcells were collected. Whole-cell lysates were prepared. Equal amounts ofproteins were subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and the polypeptides were transferred to anitrocellulose membrane. Phosphorylation of IRF3 was determined using arabbit polyclonal antibody specific for phosphoserine-396 of IRF3 (CellSignaling). The level of IRF3 was determined using a rabbit polyclonalantibody against IRF3 (Cell Signaling). Anti-STING antibodies werepurchased from Cell Signaling. Vaccinia E3 protein level was determinedby using anti-E3 monoclonal antibody (MAb 3015B2) kindly provided by Dr.Stuart N. Isaacs (University of Pennsylvania) (66) (Weaver et al. VirusRes 130: 269-274 (2007)). Anti-PARP and anti-MCL-1 antibodies (CellSignaling) were used to determine PARP cleavage and MCL-1 proteindegradation. Anti-glyceraldehyde-3-phosphate dehydrogenase (GADPH) oranti-β-actin antibodies (Cell Signaling) were used as loading controls.

Tumor Implantation and Intratumoral Injection with Viruses

B16-F10 melanoma cells (lx 10⁵) were implanted intradermally into theshaved skin on the right flank C57BL/6J mice. After 10 to 12 days postimplantation, tumor sizes were measured and tumors that were 3 mm indiameter or larger were injected with MVA or MVAΔE3L (2×10⁷ pfu) or PBSwhen the mice were under anesthesia. Viruses were injected weekly or asspecified in each experiment. Mice were monitored daily and tumor sizeswere measured twice a week. Tumor volumes were calculated according tothe following formula: l (length)×w (width)×h (height)/2. Mice wereeuthanized for signs of distress or when the diameter of the tumorreached 10 mm Serum was collected when the mice were euthanized.

MC38 murine colon adenocarcinoma cells (2×10⁵) were implantedintradermally into the shave skin on the right flank of C57BL/6J mice.After 7 days post implantation, tumor sizes were measured and tumorsthat are 2-3 mm in diameter were injected with PBS or MVAΔE3L (2×10⁷pfu) when the mice were under anesthesia. Tumor sizes were measured atvarious days post viral injection.

Bilateral Tumor Implantation Model and Intratumoral Injection with MVAor MVAΔE3L

Briefly, B16-F10 melanoma cells were implanted intradermally to the leftand right flanks of C57B/6 mice (5×10⁵ to the right flank and 1×10⁵ tothe left flank). 8 days after tumor implantation, the larger tumors onthe right flank were intratumorally injected with 2×10⁷ pfu of MVA or anequivalent amount of MVAΔE3L. The tumor sizes were measured and thetumors were re-injected twice a week. The survival of mice wasmonitored.

In some experiments, MC38 colon adenocarcinoma cells were implantedintradermally to the left and right flanks of C57B/6 mice (5×10⁵ to theright flank and 1×10⁵ to the left flank).

Flow Cytometry

To analyze immune cell phenotypes and characteristics in the tumors ortumor draining lymph nodes, we generated cell suspensions prior to FACSanalysis according to the following protocol (46) (Zamarin et al.,Science Translational Medicine 6, 226-232 (2014)). First we isolatedtumors using forceps and surgical scissors three days post treatmentwith MVA or PBS. The tumors were then weighed. Tumors or tumor draininglymph nodes were minced prior to incubation with Liberase (1.67 WunschU/ml) and DNase (0.2 mg/ml) for 30 minutes at 37° C. Cell suspensionswere generated by repeated pipetting, filtered through a 70-μm nylonfilter, and then washed with complete RPMI prior to Ficoll purificationto remove dead cells. Cells were processed for surface labeling withanti-CD3, CD45, CD4, and CD8 antibodies. Live cells are distinguishedfrom dead cells by using fixable dye eFluor506 (eBioscience). They werefurther permeabilized using FoxP3 fixation and permeabilization kit(eBioscience), and stained for Ki-67, FoxP3, and Granzyme B. For thestaining of the myeloid cell population, Fluorochromeconjugatedantibodies against CD45.2 (104), CD11b (M1/70), Ly-6C (HK1.4), MHC II(M5/114.15.2), CD24 (M1/69), F4/80 (BM8), CD103 (2E7) and CD11c (N418)were purchased from eBioscience. All antibodies were tested with theirrespective isotype controls. Data were acquired using the LSRII Flowcytometer (BD Biosciences). Data were analyzed with FlowJo software(Treestar).

Reagents

The commercial sources for reagents were as follows: CpGoligodeoxynucleotide ODN2216 (Invitrogen); cGAMP was purchased fromInvivoGen. Anti-PARP, anti-Mcl1 antibodies were obtained from CellSignaling. Antibodies used for flow cytometry were purchased fromeBioscience (CD45.2 Alexa Fluor 700, CD3 PE-Cy7, CD4 APC-efluor780, CD8PerCP-efluor710, FOXP3 Alexa Fluor 700, CD45.2 eFluor 450, CD11bAPC-eFluor 780, Ly-6C PE, MHC II PE-eFluor 610, CD24 APC, F4/80PerCP-Cy5.5, CD103 FITC, CD11c Alexa Fluor 700). Invitrogen (CD4 QDot605, Granzyme B PE-Texas Red, Granzyme B APC), BD Pharmingen(Ki-67-Alexa Fluor 488). Anti-phosphoserine-396 of IRF3 and anti-IRF3antibodies were purchased from Cell signaling.

Statistics

Two-tailed unpaired Student's t test was used for comparisons of twogroups in the studies. Survival data were analyzed by log-rank(Mantel-Cox) test. The p values deemed significant are indicated in thefigures as follows: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Tumor Infiltrating Lymphocytes (TIL) Preparation

Tumors were dissociated from mice and the total weight of tumor wasevaluated before grinded with scissors. Tumors were then digested with1.7 U/ml Liberase (Roche) and 100 μg/ml DNAse (Sigma) in RPMI 1640 andincubated at 37° C. shaker for 30 min. After digestion, the cell sampleswere diluted in RPMI 1640 and passed through a cell strainer. Next Cellswere washed with RPMI 1640 and resuspended in FACS buffer. Single cellswere kept on ice before staining for flow cytometric analysis.

Flow Cytometry of TIL

TILs were pre-incubated with 2.4G2 mAb to block FcγR binding, andstained with panels of antibodies for 30 min on ice.Fluorochrome-conjugated antibodies against CD45.2 (104), CD11b (M1/70),Ly-6C (HK1.4), MHC II (M5/114.15.2), CD24 (M1/69), F4/80 (BM8), CD103(2E7) and CD11c (N418) were purchased from eBioscience. All antibodieswere tested with their respective isotype controls. Viability wasassessed by staining with LIVE/DEAD kit (Invitrogen). All samples wereacquired with a LSRII flow cytometer (Becton Dickinson) and analyzedwith FlowJo software (Tree Star).

Protocol for the Generation of Primary Fibroblasts from Mouse Skin

Mice were euthanized by CO₂ inhalation, shaved, and chemicallydepilated. They were submerged in 70% ethanol for 1-2 min. Truncal skinswere cut out, and placed in PBS on the lid of a 100-mm tissue culturedish and spread it out with the epidermal side down. After removal ofthe subcutaneous tissue by scraping the dermal side using two pairs offorceps, skin samples were incubated with 500 μl dispase (0.5 U/ml)/PBSfor 45 min at 37° C. Skin samples were placed on the lid of a 100-mmtissue culture dish with the epidermal side up, and the epidermis wasremoved mechanically using two pairs of forceps. The dermal sheets werewashed with PBS four times before they were digested in type Icollagenase (4 mg/ml in PBS with 1% BSA) for three hours at 37° C. Cellswere cultured in DME medium with 20% FBS for 1-2 weeks.

Example 1 MVA Induces Type I IFN Production in Murine cDCs

To test whether MVA can induce type I IFN induction, bone marrow-deriveddendritic cells were cultured in the presence of GM-CSF (GM-CSF-BMDCs orcDCs) and infected with either WT VAC or MVA at a multiplicity ofinfection (MOI) of 10. Supernatants were collected at various timepoints post infection (1, 4, 8, 14, and 22 hours) and IFN-α and IFN-βprotein levels evaluated by ELISA. As shown in FIG. 1A, both IFN-α andIFN-β were detected at 8 h post-infection with MVA and continued toaccumulate up to 24 h post-infection.

To test whether WT VAC or MVA infection of cDCs affects type I IFN geneexpression, quantitative real-time PCR analysis was performed using RNAisolated from GM-CSF-cultured cDCs infected with WT VAC or MVA at 6 hpost-infection. Mock-infection controls were also included in theexperiment. As demonstrated in FIG. 1B, MVA infection of cDCs increasedIFNA4 and IFNB mRNA levels by 6-fold and 105-fold, respectively, whencompared with untreated cells. By contrast, infection with WT VACincreased IFNA4 and IFNB mRNA levels by 2-fold and 6-fold, respectively(FIG. 1B). These results indicate that MVA is a substantially strongerinducer of IFNA4 and IFNB gene expression than WT VAC (p<0.001). Thisexperiment illustrates that WT VAC and MVA have different effects on thehost's immune system (here assessed by the effect on dendritic cells)starting with the ability of MVA but not WT VAC to induce expression ofType I interferons represented by IFN-α and IFN-β.

Example 2 MVA-Induced Type I IFN Production in Murine cDCs is Dependenton IRF3/IRF7/IFNAR1

Transcription factors IRF3 and IRF7 are key regulators of type I IFNinduction and are critical for host defense against virus infections(67) (Sato et al., Immunity 13: 539-548, 2000). To test whether inMVA-induction of type I IFN requires IRF3 and IRF7, cDC were generatedfrom IRF3^(−/−), IRF7^(−/−) and WT mice (age-matched), and infected withMVA. MVA-induced IFN-α/β secretion was abolished in IRF3 deficient cDCs(FIG. 2A). IRF7^(−/−) cells fail to produce IFN-α in response to MVAinfection, while IFN-β induction was reduced by 57% in MVA-infectedIRF7^(−/−) cells (FIG. 2B).

To assess whether the type I IFN positive feedback loop mediated byIFNAR1 is required for the induction of IFN, IFNAR1^(−/−) cDCs and WTcontrols were infected with MVA at a MOI of 10 and IFN-α/β secretionslevels evaluated by ELISA. IFN-α induction by MVA was abolished inIFNAR1^(−/−) cells, whereas IFN-β induction by MVA was reduced by 45% inIFNAR1^(−/−) cells compared with WT controls (FIG. 2C). Collectively,these results indicate that: (i) IRF3 is the critical transcriptionfactor for MVA-induced type I IFN production, and (ii) IRF7 and IFNAR1play roles in amplifying type I IFN signaling induced by MVA infection.These results therefore confirm the ability of MVA to induce type I IFNvia a mechanism relevant in the activation of the immune system.

Example 3 MVA-Induced Type I IFN Induction is Dependent on STING

STING is an endoplasmic reticulum-associated protein essential for typeI IFN induction in response to intracellular DNA or DNA pathogensincluding bacteria and DNA viruses (68, 69) (Ishikawa et al. Nature 455:674-678 (2008); Barber et al. Curr Opin Immunol 23: 10-20 (2011). Totest whether STING is required for type I IFN induction in cDCs by MVA,cDCs were generated from the N-ethyl-N-nitrosourea (ENU)-inducedGoldenticket (Gt) mutant mice (Sting^(Gt/Gt)) harboring a singlenucleotide variant of Sting resulting in a functionally null allele (70)(Sauer et al. Infect Immun 79: 688-694 (2011)). cDCs from age-matched WTmice were used as a control. Cells were either infected with MVA at aMOI of 10 or treated with lipopolysaccharide (LPS). MVA induction ofIFN-α/β was abolished in Sting^(Gt/Gt) cells, whereas LPS-inducedIFN-α/β production was not affected (FIG. 3A). Induction of IFNA4 mRNAby MVA was reduced from 6-fold in WT cells to 2-fold in Sting^(Gt/Gt)cells, whereas induction of IFNB mRNA by MVA was reduced from 133 foldin WT cells to 14 fold in Sting^(Gt/Gt) cells (FIG. 3B). Furthermore,Western blot analysis demonstrated that MVA-induced IRF3 phosphorylationpeaked at 4 and 6 h post infection in WT cDCs and was absent inSting^(Gt/Gt) cDCs (FIG. 3C). Together, these results demonstrate thatSTING is essential for MVA-induced type I IFN production and IRF3phosphorylation in cDCs.

The ability of MVA to activate STING and its downstream signalingpathway including transcription factor IRF3 makes it different fromreplication competent WT VAC, which is unable to activate STING/IRF3.The inventors infer that the mechanism of MVA-mediated antitumorimmunity would be different from the mechanism of oncolytic effectsmediated by WT VAC or recombinant replication competent VAC withdeletion of thymidine kinase.

Example 4 MVA Triggers Type I IFN Production In Vivo in aSTING/IRF3-Dependent Manner

To test whether MVA triggers type I IFN production in vivo in aSTING/IRF3-dependent manner, Sting^(+/+), Sting^(Gt/Gt), and IRF3^(−/−)were infected with 2×10⁷ pfu via tail vein injection. Serum wascollected at 6 h post-infection. MVA infection of Sting^(+/+) miceinduced IFN-α and IFN-β production to the levels of 798 pg/ml and 1017pg/ml, respectively, which was abolished in Sting^(Gt/Gt), andIRF3^(−/−) mice (FIG. 3D). These results indicate that MVA-induced typeI IFN production in vivo is also dependent on STING and transcriptionfactor IRF3.

The ability of MVA to induce type I IFN production in aSTING/IRF3-dependent manner, through intravenous delivery, also pointsto IFN-dependent therapeutic effects mediated by MVA.

Example 5 cGAS is Required for the Induction of Type I IFN by MVA incDCs

The STING/IRF3 pathway can be activated by cyclic GMP-AMP (cGAMP), asecond messenger produced by cyclic GMP-AMP synthase (cGAS) in responseto DNA virus infection (71) (Sun et al. Science 339: 786-791 (2013). Totest whether MVA infection of cDCs triggers type I IFN induction via thecytosolic DNA-sensing pathway mediated by the cytosolic DNA sensor cGAS,cDCs were generated from cGAS^(−/−) (72) (Li et al., Science 341(6152):1390-1394 (2013)) mice and age-matched WT controls and infected withMVA. As shown in FIG. 4A, MVA-induced IFN-α/β production was abolishedin cGAS^(−/−) cells. Induction of IFNA4 and IFNB mRNA by MVA was alsodiminished in cGAS^(−/−) cells compared with WT cells (FIG. 4B).Finally, Western blot analysis demonstrated that MVA-inducedphosphorylation of TBK1 and IRF3 was absent in cGAS^(−/−) cells (FIG.4C). These results demonstrate that cGAS is a critical cytosolic DNAsensor for MVA.

Example 6 MVAΔE3L Induces Higher Levels of Type I IFN Production inMurine cDCs than MVA

E3 is a key virulence factor that attenuates various innate immuneresponses, including type I IFN induction. MVA retains the E3L gene.Western blot analysis showed that E3 protein was produced in WT VAC andMVA-infected BMDCs, but not in MVAΔE3L-infected cells (FIG. 5A). To testwhether E3 plays an inhibitory role in MVA sensing in cDCs, theinduction of type I IFN gene expression was compared between MVA andMVAΔE3L. It was found that MVAΔE3L induced higher levels of IFNA4 andIFNB mRNAs than MVA (FIG. 5B) (P<0.001). This induction was abolished incells lacking transcription factor IRF3 (FIG. 5C). Furthermore, Westernblot analysis demonstrated that MVAΔE3L infection induced higher levelof phospho-IRF3 than MVA at both 4 and 8 h post infection (FIG. 5D).These results suggest that E3 dampens innate immune-sensing of MVA andthat removing E3 from MVA results in enhanced activation of type I IFNgene expression. The inventors infer that MVAΔE3L may have strongerantitumor effects than MVA due to its superior ability to induce type IIFN compared with MVA.

Example 7 The Cytosolic DNA-Sensing Pathway Mediated by cGAS Plays anImportant Role in MVAΔE3L-Induced Type I IFN Induction in cDCs

To test whether MVAΔE3L infection of cDCs triggers type I IFN inductionvia the cytosolic DNA-sensing pathway mediated by the cytosolic DNAsensor cGAS (cyclic GMP-AMP synthase) (71, 73) (Sun et al., Science339(6121): 786-791 (2013); Wu et al., Science 339(6121): 826-830(2013)), and its adaptor STING (68, 74) (Ishikawa et al., Nature455(7213): 674-678 (2008); Gao et al., Cell 154(4): 748-762 (2013)),cDCs were generated from cGAS^(−/−) (72) (Li et al., Science 341(6152):1390-1394 (2013)) mice and age-matched WT controls and infected withMVAΔE3L. Using quantitative real-time PCR analysis, it was found thatMVAΔE3L-induced IFNA4 and IFN-β gene expression at 6 h post infectionwere both reduced in cGAS-deficient cells (FIG. 6A, P<0.001). ELISAanalysis of supernatants collected at 22 h post infection also showedthat MVAΔE3L-induced IFN-α/β secretion was significantly reduced incGAS-deficient cells (FIG. 6B, P<0.001). By contrast, cGAMP treatment at15 μM final concentration induced IFN-α/β secretion in both WT andcGAS^(−/−) cDCs at similar levels. Finally, Western blot analysisdemonstrated that MVAΔE3L-induced phosphorylation of IRF3 was absent at2 and 4 h post infection and significantly reduced at 6 and 8 hpost-infection in cGAS^(−/−) cells (FIG. 4C). These results demonstratethat cGAS is a critical cytosolic DNA sensor for MVAΔE3L. These resultsalso imply that the ability of MVAΔE3L to induce cGAS/STING pathway indendritic cells may contribute to its therapeutic benefits. Based onresults obtained by the present inventors with inactivated MVA anddescribed in PCT US2016/019663 filed Feb. 25, 2016, incorporated byreference in its entirety for all purposes, the inventors expect thatthe antitumor effects of MVAΔE3L will be diminished in mice that aredeficient of cGAS or STING. WT VAC with deletion of E3 is unable toinduce type I IFN in dendritic cells (data not shown), indicating thatviral inhibitor(s) of the cGAS/STING pathway expressed by WT VAC mightbe missing in MVA.

Example 8 The Cytosolic dsRNA-Sensing Pathway Mediated by MDA5/MAVS AlsoContributes MVAΔE3L-Induced Type I IFN Production in cDCs

Western blot analysis showed that MVAΔE3-induced phosphorylation of IRF3was diminished at 4 h and reduced at 8 h post infection inSTING-deficient bone marrow-derived cDCs generated from STING^(Gt/Gt)mice compared with WT cells (FIG. 7A). MVAΔE3L-induced phosphorylationof IRF3 was also reduced at 8 h post infection in MAVS^(−/−) cDCscompared with WT cells (FIG. 7A). These results suggest that MVAΔE3Linfection of cDCs could be sensed by both the cytosolic DNA-sensingpathway mediated by cGAS/STING as well as the cytosolic dsRNA-sensingpathway mediated by MDA5/MAVS. To test that hypothesis,STING^(Gt/Gt)/MDA5^(−/−) mice were generated, and double-deficient cDCsisolated. MVAΔE3L-induced phosphorylation of TBK1 and IRF3 was abolishedin STING^(Gt/Gt)/MDA5^(−/−) cells (FIG. 7B). These results indicate thatthe cytosolic dsRNA-sensing pathway also plays a role in sensing dsRNAproduced by MVAΔE3L. Taken together, these results demonstrate thatMVAΔE3L infection in cDCs leads to the cytosolic detection of viral DNAand dsRNA by the cGAS/STING and MDA5/MAVS signaling pathways,respectively, which results in the activation of TBK1 and IRF3 and theinduction of type I IFN gene expression. It has been shown thatintravenous delivery of synthetic dsRNA activates MDA5 and inducesantitumor effects (Tormo et al., 2009). The ability of MVAΔE3L to induceactivate MDA5 through the production of dsRNA in both immune cells,fibroblasts (see example) and tumor cells (see example) may alsocontribute to its therapeutic benefits.

Example 9 The Cytosolic DNA-Sensing Pathway Mediated by cGAS/STING Playsan Important Role in MVAΔE3L-Induced Ifnb Gene Expression in MurinePrimary Fibroblasts

Skin dermal fibroblasts constitute an important cell type in melanomastromal cells, contributing to melanoma progression and metastasisthrough the production of growth factors and other soluble mediators (Liet al., Oncogene 2003; Inada et al., 2015). To investigate whether skindermal fibroblasts also respond to MVA or MVAΔE3L, the inventorsgenerated primary skin fibroblasts from WT and cGAS^(−/−) mice, andinfected them with MVA or MVAΔE3L at a MOI of 10. Cells were collectedat 6 h post infection. Using quantitative real-time PCR analysis, theinventors found that MVAΔE3L infection induced higher levels of Ifnb,Ccl4, and 116 gene expression at 6 h post infection than MVA (FIG. 8A-D). The induction of Ifnb, Ccl4, Ccl5, and 116 gene expression by MVAand MVAΔE3L was diminished in cGAS-deficient fibroblasts (FIG. 8 A-D).These results demonstrate that MVA and MVAΔE3L infection of fibroblastscan be detected by the cytosolic DNA sensor cGAS, which leads to theinduction of IFNB, and other proinflammatory cytokines and chemokines.STING is a critical adaptor for the cytosolic DNA-sensing pathway. MDA5is a cytosolic dsRNA sensor (Gitlin et al., PNAS 2006). To test whetherSTING or MDA5, or both are also involved in sensing MVA or MVAΔE3L inskin fibroblasts, the inventors generated primary skin fibroblasts fromWT, STING^(Gt/Gt,) MDA5^(−/−), and STING^(Gt/Gt) MDA5^(−/−) mice, andinfected them with MVA or MVAΔE3L. Quantitative real-time PCR analysisshowed that MVA or MVAΔE3L induced Ifnb, Ccl4, Ccl5, and 116 geneexpression was largely diminished in STING-deficient cells, confirmingthat the cytosolic DNA-sensing pathway mediated by cGAS/STING iscritical for detecting viral infection and inducing an antiviral innateimmunity. There were some low residual levels of 116 and Ccl4 geneexpression induced by MVAΔE3L in STING-deficient cells, and those levelswere gone in the STING and MDA5-double deficient cells, indicating thatMDA5 plays a supporting role in detecting dsRNA produced by MVAΔE3Lvirus. The inventors conclude that in skin dermal primary fibroblasts,MVA is detected by the cGAS/STNG cytosolic DNA-sensing pathway to induceIfnb, Ccl4, Ccl5, and 116 gene expression, whereas MVAΔE3L activatesboth cGAS/STING and MDA5 pathways to induce innate immunity.

Example 10 The Cytosolic DNA-Sensing Pathway Mediated by cGAS/STINGPlays an Important Role in MVA and MVAΔE3L-Induced Type I IFN,Inflammatory Cytokine and Chemokine Production in Murine PrimaryFibroblasts

To correlate protein secretion from infected skin fibroblasts, theinventors collected supernatants from skin fibroblasts from WT,STING^(Gt/Gt), MDA5^(−/−), and STING^(Gt/Gt) MDA5^(−/−) mice infectedwith MVA or MVAΔE3L and performed ELISA analysis. It was observed thatMVAΔE3L infection induced higher secretion levels of IFN-β, IL-6 andCCL4 than MVA. Similar to what it was observed with gene expressionanalysis, MVA-induced secretion of IFN-β, IL-6 and CCL5 was abolished inSTING-deficient cells. Although MVAΔE3L-induced IFN-β and CCL5 werediminished in STING-deficient cells, there were residual levels of IL-6and CCL4 in the supernatants of STING-deficient cells infected withMVAΔE3L virus, which were abolished in the STING and MDA5-doubledeficient cells. Taken together, these results showed that skinfibroblasts are an important source for the production of IFN-β, IL-6,CCL4, and CCL5 in response to MVA or MVAΔE3L infection, which isdependent on the cytosolic DNA-sensing pathway. The inventors infer thattumor stromal fibroblasts are also capable of induction of type I IFNand proinflammatory cytokines and chemokines in response to immuneactivating viruses such as MVA or MVAΔE3L.

Example 11 MVAΔE3L Infection of B16 Melanoma Cells Induces Type I IFNand Inflammatory Cytokines/Chemokines Production

To test whether MVAΔE3L infection of B16 melanoma cells also inducestype I IFN, and inflammatory cytokine/chemokine production, B16 melanomacells were infected with MVA or MVAΔE3L at a MOI of 10, or mock-infected(NT). Cells were collected at 6 h post infection and real-time PCRanalysis was performed to analyze gene expression. As shown in FIG.10A-F, MVAΔE3L infection induced higher levels of IFNA4 (FIG. 10A), IFNb(FIG. 10B), Il6 (FIG. 10C), TNF (FIG. 10D), CCL5 (FIG. 10E) and CXCL-10(FIG. 10F) gene expression than MVA.

Example 12 Infection of B16 Melanoma Cells with Either MVA or MVAΔE3LInduces Apoptosis

Proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) by caspasesis a hallmark of apoptosis. Vaccinia E3 has been shown to inhibitapoptosis induced by dsRNA in HeLa cells (75, 76) (Kibler et al., JVirol 71, 1992-2003 (1997); Lee et al., Virology 199, 491-496 (1994)).MVAΔE3L induces more apoptosis in chicken embryonic fibroblasts (CEFs)than MVA (28) (Hornemann et al., J Virol 77, 8394-8407 (2003)). Toevaluate whether MVA and MVAΔE3L induce apoptosis in melanoma cells, B16melanoma cells were infected with either MVA or MVAΔE3L at a MOI of 10and cleavage of PARP was determined using Western blot analysis. Asshown in FIG. 9A, both MVA and MVAΔE3L infection triggered cleavage ofPARP from 116-kDa full-length protein to 89-kDa fragment at 22 h postinfection. Furthermore, MVAΔE3L induced PARP cleavage more efficientlythan MVA (FIG. 11A).

In accordance with the observation that MVA and MVAΔE3L triggerapoptosis, MVA and MVAΔE3L infection caused the degradation of Myeloidcell leukemia-1 (Mcl-1) protein, an important anti-apoptotic Bcl-2family member (FIG. 11B). Taken together, these results show thatMVAΔE3L infection of melanoma cells triggers apoptosis to the same oreven a greater extent than MVA.

Example 13 Intratumoral Injection of MVA or MVAΔE3L Leads to ProlongedSurvival of Tumor-Bearing Mice and the Generation of Systemic AntitumorImmunity

The transplantable in vivo B16 melanoma model involves the intradermalimplantation of murine B16F10 melanoma cells (1×10⁵) on one flank ofC57B/6 mice. Twelve days following tumor implantation, when the tumorswere approximately 3 mm in diameter, MVA or MVAΔE3L (2×10⁷ pfu) or PBSwas injected to the tumors weekly. Intratumoral injection of MVAresulted in tumor eradication in 60% of treated mice and prolongedsurvival in the rest (FIG. 12B-D), whereas intratumoral injection ofMVAΔE3L resulted in tumor eradication in 57% of treated mice andprolonged survival in the rest (FIG. 12B-D), demonstrating excellenttherapeutic efficacy with both agents. By contrast, all of the mice withintratumoral injection of PBS had continued tumor growth and wereeuthanized at a median of 21 days post tumor implantation (FIGS. 12A andD). In the initial experiment, intratumoral injection of MVA seemed tohave at least similar anti-tumor efficacy as MVAΔE3L virus.

To test whether mice whose tumors were eradicated after intratumoralinjection of MVA or MVAΔE3L developed systemic anti-tumoral immunity inthe animals, animals were challenged by intradermal injection of alethal dose of B16 melanoma cells (5×10⁴) to the contralateral side 8weeks after the eradication of initial tumors. Naïve mice that werenever exposed to B16 melanoma cells or viruses were used as a control inthe challenge experiment. Animals were followed for 70 days after tumorchallenge. 80% of MVA-treated mice and 100% of MVAΔE3L-treated micesurvived the tumor challenge, whereas all of the naïve mice developedgrowing tumors and were eventually euthanized (FIG. 10E). Collectively,these results suggest that intratumoral injection of MVA or MVAΔE3Lleads to tumor eradication, prolonged survival, as well as to thedevelopment of systemic antitumor immunity. In addition, this experimentindicates efficacy of the disclosed treatments in inhibiting metastasisrepresented by the tumor challenge.

Example 14 Intratumoral Injection of MVA Leads to Immunological Changesin the Tumor Environment

To investigate the immunologic changes within the tumors induced byintratumoral injection of MVA, tumors were harvested at 3 days postintratumoral injection of MVA or PBS and the immune cell infiltrateswere analyzed by FACS. We observed that the percentage of Foxp3⁺CD4⁺ Tcells (i.e., regulatory CD4+ T cells) decreased from 34.7% inPBS-treated tumors to 7.0% in MVA-treated tumors (P<0.0001, FIG. 13A-C).We also observed an increase in the percentage of CD8⁺ T cells thatexpress Granzyme B (i.e. expressing the cytotoxic phenotype) within thetumors treated with MVA (89%) compared those treated with PBS (31%)(P<0.0001, FIG. 13D-F). The percentage of Ki-67⁺CD8+ T cells (i.e.,proliferating CD8+ T cells) was increased from 51% in the PBS-treatedtumors to 76% in the MVA-treated tumors (P=0.0004, FIG. 11, G-I). Theseresults indicate that intratumoral injection of MVA dramaticallyupregulates immune responses in the tumor microenvironment, includingproliferation and activation cytotoxic CD8⁺ T cells and a concomitantreduction of CD4+ regulatory T cells within the tumors. We expect to seesimilar changes in immune cell infiltrates within tumors treated withMVAΔE3L. These experiments have been planned.

Example 15 Intratumoral Injection of MVA Also Induces ImmunologicalChanges in the Tumor-Draining Lymph Nodes (TDLNs)

To test whether intratumoral injection of MVA also causes immunologicalchanges in TDLNs, TDLNs were isolated from MVA- or PBS-injected mice atthree days post treatment, and analyzed by FACS. The percentage ofGranzyme B⁺CD8⁺ T cells in TDLNs increased from 0.15% in mice treatedwith PBS to 4.6% in mice treated with MVA (P=0.0002, FIG. 14A-C). Inaddition, the percentage of Ki-67⁺CD8⁺ T cells increased from 7.2% inmice treated with PBS to 15% in mice treated with MVA (P=0.0008, FIG.14D-F). These results indicate that there are more activated andreplicating CD8⁺ T cells in the TDLNs in MVA-treated mice than inPBS-treated mice. Taken together, these results indicate thatintratumoral injection of MVA leads to the activation and proliferationof both CD8⁺ T cells not only locally within the tumor but alsosystemically in the host.

Example 16 MVA and MVAΔE3L Infection of MC38 Murine Colon AdenocarcinomaCells Induces Type I IFN and Inflammatory Cytokines/ChemokinesProduction

To address whether MVA and MVAΔE3L also trigger similar responses inother types of solid tumor cells, the abilities of MVA and MVAΔE3L toinduce type I IFN pathway were tested in the MC38 colon adenocarcinomacells. MC38 cells were infected with MVA or MVAΔE3L at a MOI of 10, ormock-infection control. Supernatants were collected at 22 h postinfection. Using ELISA, it was determined that MVAΔE3L induced higherlevels of production of IFN-β, IL-6, CCL4 and CCL5 in MC38 cells thanMVA (FIG. 15A-D). Similarly, real-time PCR analysis revealed thatMVAΔE3L infection triggered higher levels of Ifnb, Il6, Ccl4, and Ccl5gene expression in MC38 cells than MVA (FIG. 15E-H). Western blotanalysis demonstrated that MVAΔE3L infection triggered higher levels ofphosphorylation of IRF3 than MVA in MC38 cells at 22 h post infection(FIG. 15I). These results show that the efficacy of the presenttreatment is not confined to melanoma. Moreover, because the choice ofcolon carcinoma was arbitrary and because the two tumors are notrelated. Other than both being solid tumors and of basal cell origin(carcinomas), the present results can be extrapolated to all carcinomas.Moreover, because the present inventors have shown that MVA and MVAΔE3Lexert their activity on the immune system systemically and in a tumorantigen-independent manner, the present findings can be furtherextrapolated to solid tumors.

Example 17 MVAΔE3L Infection of MC38 Murine Colon Adenocarcinoma CellsInduces Apoptosis

To investigate whether MVA and MVAΔE3L also trigger apoptosis in MC38murine colon adenocarcinoma cells, MC38 cells were infected with MVA orMVAΔE3L at a MOI of 10, or mock-infection control. As observed in theB16 melanoma cells, Western blot analysis showed that both MVA andMVAΔE3L triggered cleavage of PARP from 116-kDa full-length protein to89-kDa fragment (at 22 hours, FIG. 15I). Together, Examples 16 and 17indicate that each of MVA and MVAΔE3L has the capacity to induce type IIFN and inflammatory cytokines/chemokines production, as well asapoptosis in different types of cancer cells. MVAΔE3L seems to be astronger inducer of both type I IFN and proinflammatory cytokine andchemokine production, as well as apoptosis compared with MVA.Nevertheless, both results indicate that the immune response elicited bythe present viruses carries through to apoptosis, resulting in cancercell death. This further establishes the presently disclosed treatmentsas a viable approach to therapy of melanoma, colon cancer, carcinomas ingeneral and indeed solid tumors.

Example 18 MVAΔE3L Inhibits Tumorigenesis in Murine Model of ColonCarcinoma

Experimental studies disclosed in Example 11 showed that intratumoralinjection of MVA or MVAΔE3L leads to tumor eradication and systemicanti-tumoral immunity in a murine transplantable B16 melanoma model. Totest whether MVAΔE3L is capable of inhibiting tumor growth in othersolid tumors, the inventors tested the anti-tumor effects of MVAΔE3L ina murine colon carcinoma implantation model. Colon carcinoma isrepresentative of a tumor not related to melanoma but was otherwise arandom choice. 2×10⁵ MC38 colon carcinoma cells were intradermallyimplanted into the right flank of C57B/6 mice. Tumors were allowed toform for 7 days, after which MVAΔE3L (2×10⁷) or PBS control wereintratumorally injected into mice. Tumors were measured at prior toinjection (day 0) and for up to 45 days post injection and tumor volumewas calculated according the following formula: l (length)×w (width)×h(height)/2. As shown in FIGS. 16 (A and B), tumors treated with MVAΔE3Lwere significantly smaller than PBS-treated tumors. Furthermore, micetreated with MVAΔE3L exhibited improved survival as demonstrated by theKaplan-Meier survival curve of tumor-bearing mice injected with PBS orMVAΔE3L (FIG. 16C). Collectively, these findings reveal that in thecontext of colon cancer as well as melanoma, MVAΔE3L maintains thecapacity to inhibit tumorigenesis and tumor growth. Collectively,results observed here as well as in Example 13 and Example 17demonstrate and illustrate that MVAΔE3L is efficient in promotinganti-tumor effects in various solid tumors and that the findingsdescribed in this disclosure are not limited to melanoma but can beextrapolated to other solid tumors of diverse origins.

Example 19 Intratumoral Injection of MVA and MVAΔE3L Leads to AntitumorEffects in Both Injected and Non-Injected Distant Tumors and EnhancedSurvival

Next, the inventors investigated the effects of intratumoral injectionof MVA and MVAΔE3L on metastatic growth using a murine B16-F10 melanomabilateral implantation model. Briefly, B16-F10 melanoma cells wereimplanted intradermally to the left and right flanks of C57B/6 mice(5×10⁵ to the right flank and 1×10⁵ to the left flank). 8 days aftertumor implantation, the inventors intratumorally injected MVA or MVAΔE3L(2×10⁷ pfu) or PBS to the larger tumors on the right flank twice weekly.The tumor sizes were measured and the survival of mice was monitored(FIG. 17). Whereas the PBS-treated mice died quickly with increasingtumor growth over the next 10-14 days (FIGS. 17A and B), mice treatedwith MVA and MVAΔE3L exhibited delayed tumor growth of both the injectedand non-injected tumors at the contralateral side (FIG. 17A-F).

The ability to control the growth of non-injected distant tumorscorrelated with the improved survival of animals treated with MVA andMVAΔE3L (FIG. 17G, ***, P<0.001 for MVA vs. PBS, ****, P<0.0001 forMVAΔE3L vs. PBS). Collectively, these results illustrate the soundnessof a therapeutic approach for the treatment of metastatic solid tumors.

Example 20 Intratumoral Injection of MVA and MVAΔE3L Leads toImmunological Changes in Both Injected and Non-Injected Distant Tumors

To understand the immune mechanisms underlying the antitumor effects ofMVA and MVAΔE3L, the inventors investigated the immune cell infiltratesin both injected and non-injected tumors in MVA or MVAΔE3L-treated micecompared with PBS control. Briefly, 2.5×10⁵ B16-F10 melanoma cells wereintradermally implanted to the left flank and 5×10⁵ B16-F10 melanomacells to the right flank of the mice. 7 days post implantation, 2×10⁷pfu of MVA, or MVAΔE3L, or PBS were injected into the larger tumors onthe right flank. The injection was repeated three days later. Thenon-injected tumors were harvested and cell suspensions were generated.The live immune cell infiltrates in the tumors were analyzed by flowcytometry (FACS). The inventors observed a dramatic increase of CD8⁺CD3⁺immune cells in both injected and non-injected tumors of mice treatedwith MVA or MVAΔE3L compared with those in mice treated with PBS (FIGS.18 A and B). MVAΔE3L is more effective than MVA in the recruitment ofCD8⁺CD3⁺ in both injected and non-injected tumors (FIGS. 18 A and B).MVA or MVAΔE3L-treatment resulted in the increase of numbers of GranzymeB expressing CD8⁺ and CD4⁺ T cells in both injected and non-injectedtumors (FIGS. 18 C, D, I, and J). In addition, MVA or MVAΔE3L-treatmentresulted in the proliferation of effector CD8⁺ and CD4⁺ T cells in bothinjected and non-injected tumors as measured by the expression ofproliferation marker Ki-67 (FIGS. 18 E, F, K, and L). Finally,intratumoral injection of MVA and MVAΔE3L resulted in the reduction ofthe percent of FoxP3-expressing CD4⁺ T cells in the injected andnon-injected tumors (FIGS. 18 G and H). These results indicate that bothMVA and MVAΔE3L are capable of the recruitment, activation, andinduction of proliferation of effector CD8⁺ and CD4⁺′ as well as thereduction of the immune suppressive regulatory CD4⁺ T cells in theinjected and non-injected tumors. This correlates with their efficaciesin eradicating or delaying the growth of injected and non-injectedtumors and prolongation of survival. In most cases, MVAΔE3L is morepotent than MVA in the induction of immunological changes withininjected and non-injected tumors. This could be due to the enhancedabilities of MVAΔE3L in the induction of type I IFN in immune cells,tumor cells, as well as stromal cells compared with MVA.

Example 21 Intratumoral Injection of MVA and MVAΔE3L Leads to DramaticReduction of Tumor-Associated Macrophages in the Injected Tumors

To investigate whether tumor-associated macrophages (TAM) in melanomatumors were influenced by MVA or MVAΔE3L therapy, the inventors appliedpanels of antibodies to define TAM based on the strategy reportedpreviously (Broz et al., Cancer Cell 2014). Generally, live CD45⁺ andLy6C⁻ cells were first defined. Within MHCII⁺ cells, macrophages weredistinguished from DCs based on CD24^(hi) and F4/80^(lo) expression. TheTAM populations further revealed two types of macrophages (TAM1 andTAM2) by differential expression of CD11c and CD11b. After MVA orMVAΔE3L therapy, TAM population was decreased in melanoma tumor comparedwith control (FIGS. 19A and B). Meanwhile, both TAM1 and TAM2populations were declined to low levels (FIGS. 19A, C, and D). Theseobservations indicate that intratumor injection of MVA and MVAΔE3L leadsto reduction of TAMs, which induce immune suppressive effects withintumor microenvironment.

Prophetic Example 22 The Combination of Intratumoral Injection of MVA orMVAΔE3L with Intraperitoneal Delivery of Immune Checkpoint BlockadeAntibody in a Unilateral Melanoma Implantation Model

Intratumoral injection of the present viruses will be used to enhancetherapeutic effects of current immunotherapies, such as the blockade ofimmune checkpoints (for example, anti-CTLA-4 antibody), tumor-bearingmice will be treated with intratumoral injection of MVA or MVAΔE3L incombination with intraperitoneal delivery of anti-CTLA-4 antibody.Briefly, B16-F10 melanoma cells (2×10⁵) will be implanted intradermallyinto the right flank of WT C57B/6 mice. Ten days following tumorimplantation, when the tumors have grown larger than those in Example11, mice will be treated with the following combinations: PBS+isotypecontrol, PBS+anti-CTLA-4 antibody, MVA+isotype control, MVA+anti-CTLA-4,MVAΔE3L+isotype control, and MVAΔE3L+anti-CTLA. The inventors willensure that the tumor volume is consistent among tested groups at thestart of the virus injections. It is anticipated that the treatment withMVA and anti-CTLA-4 antibody, or MVAΔE3L and anti-CTLA-4 antibody willlead to superior therapeutic efficacy compared to either immunecheckpoint blockade alone or MVA or MVAΔE3L treatment alone.

Prophetic Example 23 The Combination of Intratumoral Injection of MVA orMVAΔE3L with Intraperitoneal Delivery of Immune Checkpoint Blockade in aBilateral Melanoma Implantation Model

The inventors will also intratumorally inject MVA or MVAΔE3L enhancestherapeutic effects of immune checkpoint blockade therapy such asanti-CTLA-4, anti-PD-1, or anti-PD-L1 antibodies in a bilateral B16-F10melanoma model, which also simulates an individual with metastaticdisease. Briefly, B16-F10 melanoma cells will be implanted intradermallyto the left and right flanks of C57B/6 mice (5×10⁵ to the right flankand 1×10⁵ to the left flank). 8 days after tumor implantation, MVA orMVAΔE3L will be intratumorally injected (2×10⁷ pfu of MVA or MVAΔE3L) orPBS to the larger tumors on the right flank twice weekly. Four groups ofmice were treated with MVA and four groups with MVAΔE3L, with each groupreceiving intraperitoneal delivery of either isotype control, oranti-CTLA-4, or anti-PD-1, or anti-PD-L1 antibodies.

The inventors anticipate that the combination of intratumoral injectionof MVA or MVAΔE3L and systemic delivery of checkpoint inhibitors(represented by anti-CTLcomA-4, anti-PD-1 and anti-PD-L1 antibodies)will further delay growth or eradicate the non-injected tumors comparedto intratumoral injection of either checkpoint inhibitor alone or MVA orMVAΔE3L alone.

It is anticipated that the results will show that intratumoral deliveryof MVA or MVAΔE3L overcomes treatment resistance to immune checkpointblockade in a metastatic B16 melanoma model which portends well fortransferring this approach to human therapy with beneficial results.

Prophetic Example 24 Combination of Intratumoral Injection of MVA orMVAΔE3L with Intraperitoneal Delivery of Immune Checkpoint Blockade in aBilateral MC38 Colon Adenocarcinoma Implantation Model

The inventors will further perform experiments involving intratumoralinjection of MVA or MVAΔE3L enhances therapeutic effects of immunecheckpoint blockade therapy such as anti-CTLA-4, anti- or anti-PD-L1antibodies in other bilateral tumor implantation model, which simulatesan individual with metastatic disease. Briefly, MC38 colonadenocarcinoma cells will be implanted intradermally to the left andright flanks of C57B/6 mice (5×10⁵ to the right flank and 1×10⁵ to theleft flank). 8 days after tumor implantation, MVA or MVAΔE3L will beintratumorally injected (2×10⁷ pfu of MVA or MVAΔE3L) or PBS to thelarger tumors on the right flank twice weekly. Three groups of mice willbe treated with PBS, with each group receiving intraperitoneal deliveryof isotype control, or anti-CTLA-4, or anti-PD-L1 antibodies. There willbe additional three groups of mice that will be treated with MVA, witheach group receiving intraperitoneal delivery of either isotype control,or anti-CTLA-4, or anti-PD-L1 antibodies. Finally, mice treated withMVAΔE3L will be divided into three groups, with each group treated witheither isotype control, or anti-CTLA-4, or anti-PD-L1 antibodies. Eachgroup will then be divided into a subgroup also treated with MVA orMVAΔE3L. Controls treated with virus alone will also be provided.

Tumor volume of both injected and non-injected tumors of each group ofmice will be monitored and evaluated. Additionally, the inventors willmonitor the survival of each treatment group.

It is anticipated that the combination of intratumoral delivery of MVAor MVAΔE3L with checkpoint blockade represented by intraperitonealdelivery of anti-CTLA-4 antibody or intratumoral delivery of MVA orMVAΔE3L with intraperitoneal delivery of anti-PD-1/PD-L1 will lead toeradication of non-injected distant tumors at a higher efficiency thanMVA or MVAΔE3L. Thus, it is anticipated that these results showimprovement to the treatment of metastatic solid tumors using acombination of MVA or MVAΔE3L and immune checkpoint blockade compared toeither checkpoint blockade alone or virus alone. More specifically, itis anticipated that both injected and noninjected tumors will be reducedin size and even eradicated to a degree greater than that achieved witheither type of monotherapy and that the results will persist for atleast 45 days an longer, thereby validating the combination approach forprimary and metastatic solid tumor treatment.

Prophetic Example 25 Combination of Intratumoral Injection of MVA orMVAΔE3L with Intratumoral Delivery of Immune Checkpoint BlockadeAnti-CTLA-4 Antibody in a Bilateral B16-F10 Implantation Model

In Prophetic Examples 22, 23, and 24, the inventors will test thecombination of intratumoral injection of MVA or MVAΔE3L with systemicdelivery of immune checkpoint blockade in both melanoma and colonadenocarcinoma models. In this Example, the inventors will test whetherthe co-administration of MVA or MVAΔE3L and checkpoint blockaderepresented by anti-CTLA-4 antibody (at 1/10 of dose used forintraperitoneal delivery) will achieve antitumor effects in a stringentbilateral tumor implantation model. Briefly, B16-F10 melanoma cells willbe implanted intradermally to the left and right flanks of C57B/6 mice(5×10⁵ to the right flank and 1×10⁵ to the left flank). 8 days aftertumor implantation, MVA or MVAΔE3L will be intratumorally injected(2×10⁷ pfu of MVA or MVAΔE3L or PBS) into the larger tumors on the rightflank twice weekly. Three groups of mice will be treated with MVA, witheach group receiving: (i) intraperitoneal delivery of anti-CTLA-4 (100μg/mouse) (ii) intratumoral delivery of isotype antibody (10 μg/mouse),or (iii) intratumoral delivery of anti-CTLA-4 antibody (10 μg/mouse).Additional three groups of mice will be treated with MVAΔE3L, with eachgroup receiving: (i) intraperitoneal delivery of anti-CTLA-4 (100μg/mouse) (ii) intratumoral delivery of isotype antibody (10 μg/mouse),or (iii) intratumoral delivery of anti-CTLA-4 antibody (10 μg/mouse).

Tumor volumes of both injected and non-injected tumors will be monitoredand evaluated. The inventors anticipate that the intratumoralco-injection of MVA or MVAΔE3L and checkpoint blockade (anti-CTLA-4antibody at 10 μg/mouse) will be comparable to the therapeutic effectsof the combination of intratumoral injection of MVA or MVAΔE3L andintraperitoneal delivery of anti-CTLA-4 antibody (100 μg/mouse). It isanticipated that co-administration of MVA or MVAΔE3L and an immunecheckpoint blockade at a substantially lower dose can achieve similarsystemic antitumor effects to the combination of intratumoral deliveryof MVA or MVAΔE3L with systemic delivery of anti-CTLA-4 antibody at ahigher dose.

The above specification, examples and data provide a completedescription of the manufacture and use of the composition of theinvention. However, these are illustrative and nonlimiting. The breadthof the present invention resides in the claims.

All patent and literature documents cited herein are incorporated byreference in their entirety for all purposes. Any embodiment or claimfeature disclosed herein can be disclaimed in Applicant's discretion.

What is claimed is:
 1. A method for treating a subject afflicted with amalignant solid tumor r, the method comprising delivering to the cellsof the tumor a modified vaccinia virus selected from the group of MVAand MVAΔE3Land combinations thereof and thereby treating the tumor. 2.The method of claim 1 wherein the amount of said virus is effective tobring about one or more of the following: a. induce the immune system ofthe subject to mount an immune response against the tumor or enhance anongoing response by the immune system against the tumor; b. reduce thesize of the tumor; c. eradicate the tumor; d. inhibit growth of thetumor; e. inhibit metastasis of the tumor; and f. reduce or eradicatemetastatic tumor.
 3. The method of claim 2 wherein the immune responseagainst the tumor accomplishes one or more of the following: a. increaseat least one of antitumor cytotoxic CD8⁺ T cells and effector CD4⁺ Tcells within the tumor and/or in tumor-draining lymph nodes; b. inducematuration of dendritic cells infiltrating said tumor through inductionof type I IFN; c. reduce immune suppressive (regulatory) CD4⁺ T cellswithin the tumor; d. reduce immune suppressive tumor-associatedmacrophages (TAMs) within the tumor; e. induce type I IFN, inflammatorycytokine and chemokine production in immune cells and stromalfibroblasts.
 4. The method of claim 1-3 wherein said MVA or MVAΔE3L isnot harboring nucleic acid encoding or expressing a tumor antigen. 5.The method of claim 4 wherein the tumor includes tumor located at thesite of MVA or MVAΔE3L or tumor located elsewhere in the body of thesubject.
 6. The method of claim 5 wherein the recruitment and activationof CD4⁺ effector T cells is accompanied by a reduction of regulatoryCD4⁺ cells in said tumor.
 7. The method of claim 5 wherein the tumor ismelanoma or colon carcinoma or another solid tumor.
 8. The method ofclaim 5 wherein delivery of the MVA or MVAΔE3L is continued until itinduces tumor regression or eradication.
 9. The method of one or more ofclaims 5-8 wherein delivery of the MVA or MVAΔE3L is continued forseveral weeks, months or years or indefinitely as long as benefitspersist or a maximum tolerated dose is reached.
 10. The method of claim5 wherein delivery of the MVA or MVAΔE3L is continued indefinitely untilthe maximum tolerated dose is reached.
 11. The method of one or more ofclaims 5-10 wherein delivery of the MVA or MVAΔE3L is by parenteralinjection.
 12. The method of claim 11 wherein delivery of the MVA orMVAΔE3L is by intratumoral injection.
 13. The method of claim 11 whereindelivery of the MVA or MVAΔE3L is by intravenous injection.
 14. Themethod of any one of claims 5-13 wherein the subject is a human.
 15. Themethod of one or more of claims 5-14 wherein the MVA or MVAΔE3L isdelivered at a dosage per administration within the range of about10⁵-10¹⁰ plaque-forming units (pfu).
 16. The method of claim 5-14wherein the MVA or MVAΔE3L is delivered at a dosage per administrationwithin the range of about 10⁶ to about 10⁹ plaque-forming units (pfu).17. The method of any one of the preceding claims wherein the amountdelivered is sufficient to infect all tumor cells.
 18. The method of oneor more of claims 5-17 wherein the delivery is repeated with a frequencywithin the range from once per month to two times per week.
 19. Themethod of claim 5-18 wherein the delivery is repeated once weekly. 20.The method of claim 5-19 wherein the melanoma is metastatic melanoma.21. The method of any one or more of claims 1-20 wherein the MVA isMVAΔE3L.
 22. A method for treating a solid malignant tumor in a subjectcomprising delivering to a tumor of the subject an amount of modifiedvaccinia virus Ankara (MVA) or MVAΔE3L or a combination of botheffective to bring about at least one of the following immunologiceffects: a. increase at least one of effector CD8⁺ T cells and effectorCD4⁺ T cells within the tumor and/or in tumor-draining lymph nodes; b.induce maturation of dendritic cells infiltrating said tumor throughinduction of type I IFN; c. reduce immune suppressive (regulatory) CD4⁺T cells within the tumor; d. reduce immune suppressive tumor-associatedmacrophages (TAM) within the tumor; e. induce type I IFN, inflammatorycytokine and chemokine production in immune cells and stromalfibroblasts.
 23. The method of claim 22 wherein the MVAΔE3L inducesmaturation of dendritic cells infiltrating said tumor through inductionof type I IFN.
 24. The method of claim 22 wherein the amount iseffective to recruit and activate CD4⁺ effector T cells in the subjectrecognizing cells of the tumor.
 25. The method of any one of claims22-24 wherein the recruitment and activation of CD4⁺ effector T cells isaccompanied by a reduction of regulatory CD4⁺ cells in said tumor. 26.The method of claim 25 wherein the tumor is melanoma or colon carcinomaor another solid tumor.
 27. The method of claim 25 wherein the MVA orMVAΔE3L induces type I interferon in infected tumor cells.
 28. Themethod of claim 25 wherein delivery of the MVA or MVAΔE3L is continueduntil it induces tumor regression or eradication.
 29. The method ofclaim 25 wherein delivery of the MVA or MVAΔE3L is continued as long asbenefits persist.
 30. The method of claim 25 wherein delivery of the MVAor MVAΔE3L is continued until the maximum tolerated dose is reached. 31.The method of claim 25 wherein delivery of the MVA or MVAΔE3L is byintratumoral or intravenous injection or in the case of intraperitonealmetastasis by intraperitoneal injection.
 32. The method of claim 25wherein delivery of the MVA or MVAΔE3L is by intratumoral injection. 33.The method of claim 25 wherein delivery of the MVA or MVAΔE3L is byintravenous injection.
 34. The method of claim 22, 23 24 or 25 whereinthe subject is a human.
 35. The method of claim 25 wherein the MVA orMVAΔE3L is delivered at a dosage per administration within the range ofabout 10⁵-10¹⁰ plaque-forming units (pfu).
 36. The method of claim 25wherein the MVA or MVAΔE3L is delivered at a dosage per administrationwithin the range of about 10⁶ to about 10⁹ plaque-forming units (pfu).37. The method of claim 25 wherein the amount delivered is sufficient toinfect all tumor cells.
 38. The method of claim 25 wherein the deliveryis repeated with a frequency within the range from once per month to twotimes per week.
 39. The method of claim 25 wherein the delivery isrepeated once weekly.
 40. The method of any one of claims 25-39 whereinthe melanoma is metastatic melanoma.
 41. The method of one or more ofclaims 22-40 wherein the virus is MVAΔE3L
 42. A method for treating asolid malignant tumor in a subject comprising delivering to tumor cellsof the subject an amount of MVA or MVAΔE3L or a combination thereofeffective to induce the immune system of the subject to mount an immuneresponse against the tumor or to enhance an ongoing immune response ofsaid subject against the tumor, so as to accomplish one or more of thefollowing: reduce the size of the tumor, eradicate the tumor, inhibitgrowth of the tumor, inhibit metastatic growth of the tumor, induceapoptosis of tumor cells or prolong survival of the subject.
 43. Amethod for treating a malignant tumor in a subject, the methodcomprising delivering to tumor cells of the subject a virus selectedfrom the group consisting of modified vaccinia Ankara (MVA), MVAΔE3L anda combination thereof in an amount effective to induce the immune systemof the subject to mount an immune response against the tumor or toenhance an ongoing immune response f said subject against the tumor andconjointly administering to the subject a second amount of an immunecheckpoint blocking agent or an immune checkpoint agonist effective toblock immune suppressive mechanisms within the tumor.
 44. The method ofclaim 43 wherein the immune suppressive mechanisms are elicited by tumorcells, stromal cells, or tumor infiltrating immune cells.
 45. The methodof claim 43 wherein the administration is by parenteral route.
 46. Themethod of claim 43 wherein the delivery is by intratumoral injection andthe administration is by intravenous route.
 47. The method of claim 43wherein both the delivery and the administration are by intravenousroute.
 48. The method of claim 43 wherein both the delivery and theadministration are by intratumoral injection.
 49. The method of claim 43wherein the immune checkpoint blocking agent is selected from the groupconsisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA4 inhibitors,inhibitory antibodies against LAG-3 (lymphocyte activation gene 3), TIM3(T cell Immunoglobulin and Mucin-3), B7-H3, and TIGIT (T-cellimmunoreceptor with Ig and ITIM domains); and the immune checkpointagonist is selected from the group consisting of anti-ICOS antibodyanti-OX40 antibody agonist antibody against 4-1BB (CD137) and againstGITR.
 50. The method of claim 49 wherein any one of said inhibitors isan antibody.
 51. The method of claim 43 wherein the tumor is primary ormetastatic melanoma or primary or metastatic colon carcinoma.
 52. Themethod of claim 43-46 wherein the virus is delivered and the immunecheckpoint blocking agent is administered each according to its ownadministration schedule of spaced apart intervals.
 53. The method ofclaim 52 wherein a first dose of the virus is delivered first and aftera lapse of time a first dose of the immune checkpoint blocking agent isadministered.
 54. The method of claim 52 or 53 wherein the delivery andadministration occur in parallel during the same overall period of time.55. The method of claim 52 wherein one or both of the virus and theimmune checkpoint blocking agent are respectively delivered andadministered during a period of time of several weeks, months or years,or indefinitely as long as benefits persist and a maximum tolerated doseis not reached.
 56. The method of one or more of claims 43-55 whereinthe virus is delivered at a dosage per administration within the rangeof about 10⁵-10¹⁰ plaque-forming units (pfu).
 57. The method of claim 56wherein the virus is delivered at a dosage per administration within therange of about 10⁶ to about 10⁹ plaque-forming units (pfu).
 58. Themethod of any one of claims 43-57 wherein the virus delivery is repeatedwith a frequency within the range from once per month to two times perweek.
 59. The method of claim 58 wherein the virus delivery is repeatedonce weekly.
 60. The method of one or more of claims 43-59 wherein thevirus is MVAΔE3L.
 61. The method of any one of claims 43-59 wherein thevirus is MVA.
 62. The method of one or more of claims 43-59 wherein thevirus and the immune checkpoint blocking agent are administeredsimultaneously.
 63. The method of claim 61 wherein the virus and theimmune checkpoint blocking agent are administered in the samecomposition.
 64. The method of claim 43-63 wherein the inactivated MVAand the immune checkpoint blocking agent are delivered intratumorally.65. The method of claim 43 wherein the virus and the immune checkpointblocking agent are administered sequentially.
 66. The method of claim 65wherein the inactivated MVA and the immune checkpoint blocking agent aredelivered intratumorally.
 67. A composition for use in treating a solidtumor comprising an amount of a modified vaccinia virus selected fromthe group consisting of MVA and MVAΔE3L and combinations thereofeffective to induce the immune system of a host to whom said compositionwill be administered to mount an immune response against the tumor or toenhance an ongoing immune response of the host against the tumor; and apharmaceutically acceptable carrier or diluent.
 68. The composition ofclaim 67 wherein the effective amount is within the range of about10⁵-10¹⁰ plaque-forming units (pfu) in a unit dosage form.
 69. Thecomposition of claim 68 wherein the effective amount is within the rangeof 10⁶ to 10⁹ pfu.